WO2010008764A1 - Pseudomonas fluorescens strains for production of extracellular recombinant protein - Google Patents

Abstract

Provided herein are compositions and methods for improving secretion of heterologous protein from a population of Pseudomonas fluorescens-derived cells. The compositions include host cell populations that have been genetically modified to increase secretion into the extracellular space of a heterologous protein of interest. The genetically-modified host cell populations are useful for improving the extracellular secretion of any protein of interest, including therapeutic proteins, hormones, a growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like. In various embodiments, the modified P. fluorescens host cell populations include one or more genomic mutations responsible for or contributing to the improved level of extracellular secretion. The host cell populations of the invention include the modified P. fluorescens strains deposited in the American Type Tissue Culture Collection and assigned accession numbers PTA-8981 and PTA-8982.

Description

PSEUDOMONAS FLUORESCENS STRAINS FOR PRODUCTION OF EXTRACELLULAR RECOMBINANT PROTEIN

FIELD OF THE INVENTION

This invention is in the field of protein production, particularly to identifying optimal host cells for large-scale production of properly processed heterologous proteins.

BACKGROUND OF THE INVENTION

More than 150 recombinantly produced proteins and polypeptides have been approved by the U.S. Food and Drug Administration (FDA) for use as biotechnology drugs and vaccines, with another 370 in clinical trials. Unlike small molecule therapeutics that are produced through chemical synthesis, proteins and polypeptides are most efficiently produced in living cells. However, current methods of production of recombinant proteins in bacteria often produce improperly folded, aggregated or inactive proteins, and many types of proteins require secondary modifications that are inefficiently achieved using known methods.

Numerous attempts have been developed to increase production of properly folded proteins in recombinant systems. For example, investigators have changed fermentation conditions (Schein (1989) Bio/Technology, 7:1141-1149), varied promoter strength, or used overexpressed chaperone proteins (Hockney (1994) Trends Biotechnol. 12:456-463), which can help prevent the formation of inclusion bodies. Other strategies have been developed to excrete proteins from the cell into the supernatant, including the use of various secretion signals to facilitate extracellular targeting.

Heterologous protein production often leads to the formation of insoluble or improperly folded proteins, which are difficult to recover and may be inactive.

Furthermore, the presence of specific host cell proteases may degrade the protein of interest and thus reduce the final yield. There is no single factor that will improve the production of all heterologous proteins. As a result, there is a need in the art for identifying improved large-scale expression systems capable of secreting and properly processing recombinant polypeptides to produce transgenic proteins in properly processed form.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for improving secretion of heterologous protein from a population of Pseudomonas fluorescens- derived cells. The compositions comprise host cell populations that have been genetically modified to increase secretion into the extracellular space of a heterologous protein of interest. The genetically-modified host cell populations are useful for improving the extracellular secretion of any protein of interest, including therapeutic proteins, hormones, a growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like. In various embodiments, the modified P. fluorescens host cell populations comprise one or more genomic mutations responsible for or contributing to the improved level of extracellular secretion. The host cell populations of the invention include the modified P. fluorescens strains deposited on February 27, 2008 in the American Type Tissue Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110- 2209, and assigned accession numbers PTA-8981 and PTA-8982.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic of the mutant library isolate evaluation scheme.

Figure 2 is a representative data set from one 96-well plate culture in the HTP mutant screening. Each well was inoculated with a single mutant library colony and measured for optical density (OD600) and cell-free broth PhoA activity (RFU/1000). Control strains were grown in wells Al and A2 as described in the figure. Mutant isolates exhibiting 3-fold or better activity compared to the controls, such as wells F8 and Hl 1, were noted as candidates for future evaluations. Isolates exhibiting high activity and low optical density, such as C5, were disregarded since the high activity could be attributed to cell lysis and release of intracellular PhoA.

Figure 3 shows the HTP 96-well growth and cell-free broth alkaline phosphatase activity assay results for EMS mutant strains P5-H11, P6-B6 and P23- AlO cured for plasmid DNA then re-transformed with the pDOW2299 (PhoA) expression plasmid. The white bars represent OD600 measurements of cultures 24 hours after IPTG induction (124) while the black bars represent relative fluorescent unit measurements (RFU/1000) of the filtered, cell-free broth collected. The DC454, or wild-type (WT), non-EMS treated strain harboring the pDOW2299 plasmid resides in wells A1-A4. The negative control strain DC454 harboring the "empty" plasmid pDOWl 169 (WT Vec) resides in wells A5-A8. Each of the EMS mutant parent strains were labeled as P5-H11, P6-B6 and P23-A10. The remaining cured and re- transformed isolates (e.g. 5 #1-1) were designated by cured isolate (5 #1) number and by trans formant isolate (5 # 1 - 1 ) number.

Figure 4 shows PhoA yields plotted from before induction (Hr=O) to 24 hours post-induction (Hr=24) as determined by CGE analysis of 2OL culture sample preparations. The top panel shows a plot of total culture soluble PhoA protein in g/L on the X-axis versus hours after induction on the Y-axis. The bottom panel shows a plot of PhoA protein detected in culture cell-free broth samples (CFB) in g/L on the X-axis versus hours after induction on the Y-axis. The yield data plotted for each strain identified in both figures is the calculated average for all tank runs; the standard error is indicated by the error bars.

Figure 5 is a summary bar plot of alkaline phosphatase (PhoA) yields at 124 from whole broth and cell-free broth for each mutant strain examined at 2OL scale. For each strain identified on the X-axis, the black bar indicates cell-free broth PhoA protein yield (g/L) and the grey bar indicates whole culture PhoA protein yield (g/L). When error bars are exhibited, bar graphs indicate average yield for all tank runs and error bars indicate standard deviation. Figure 6 shows the growth of P. fluorescens DC831 EMS mutant strain

(DC 825 parent cured of pDOW2299) harboring the Gal 13 diabody expression plasmid pDOW3802. The DC694 strain is the DC454/ pDOW3802 control strain that was not subjected to EMS mutagenesis. Culture absorbance (A575) is shown on the Y-axis while the elapsed fermentation time (EFT) in hours is shown on the X-axis. The run date, 2OL tank and glycerol supplier are identified in the legend for each strain.

Figure 7 shows the Gal 13 diabody yields plotted from before induction (Hr=O) to 24 hours post-induction (Hr=24) as determined by CGE analysis of 2OL culture sample preparations. The top panel shows a plot of total culture soluble Gal 13 diabody protein (g/L) on the X-axis versus hours after induction on the Y-axis. The bottom panel shows a plot of Gal 13 diabody protein detected in culture cell- free broth samples (CFB) in g/L on the X-axis versus hours after induction on the Y-axis. The yield data plotted for each strain is the calculated average for all tank runs; the standard error is indicated by the error bars.

DETAILED DESCRIPTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings.

Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Overview Bacterial expression systems are commonly used for producing large amounts of recombinant protein. The recombinant protein may be recovered from the bacterial cells or from the culture broth of the cells. One approach for recovering substantial yields of soluble and active recombinant protein from Gram-negative bacteria is to construct signal peptide fusions which direct the product to the more oxidizing environment of the periplasmic space where correct folding and disulfide bond formation are promoted. Another strategy for improving active protein yield is to promote extracellular production of recombinant protein by secreting or releasing protein localized in the periplasm into the extracellular medium. Extracellular production of recombinant protein would eliminate the need for cell disruption for protein purification, enable continuous fermentation and facilitate additional screening methods for protein yield improvements. Various strategies for the extracellular production of recombinant protein from E. coli have been previously developed, including increasing the permeability of the outer membrane by chemical and enzymatic treatments, co-overexpressing specific lysis proteins (including KiI and BRP) and using outer membrane protein fusion partners (Devoe and Gilchrist (1973) J. Exp. Med. 138, 1156-1167; Yokoyama et al. (2000) FEMS Microbiol. Lett. 192, 139-144; and, Kolling and Matthews (1999) Appl. Environ. Microbiol. 65, 1843- 1848).

Improved host cell populations

Provided herein are multiple Pseudomonas fluorescens-άeήved cell populations that result in an increase in the level of extracellular secretion of heterologous protein compared to the increase in extracellular secretion by a wildtype P.fluorescens strain. "Heterologous," "heterologously expressed," or "recombinant" generally refers to a gene or protein that is not endogenous to the host cell or is not endogenous to the location in the native genome in which it is present, and has been added to the cell by infection, trans fection, transformation, microinjection, electroporation, microprojection, or the like. A "Pseudomonas fluorescens -derived cell" is a cell that has been modified to introduce genomic changes in a Pseudomonas fluorescens cell. The genomic changes may be introduced by way of any number of mutagenesis or genetic engineering strategies known in the art. A "wildtype P. fluorescens strain" is a P. fluorescens cell that has not been genetically modified to improve the secretion of a heterologous protein of interest (for example, the Pseudomonas fluorescens strain DC454 described in Example 1).

A subset of the improved Pseudomonas fluorescens-derived strains described herein were deposited in the permanent collection of the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, on February 27, 2008, and assigned Accession Nos. PTA-8981 and PTA-8982, for strains DC847 and DC831, respectively. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Access to these deposits will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicants will make available to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit with the ATCC. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S. C. §112. The P. fluorescens-dsήyed host cell populations of the invention secrete a greater proportion of recombinant protein into the extracellular space compared to the level of secretion from a control cell population. In various embodiments, the increase in extracellular secretion is attributable to one or more genomic alterations in the improved cell population in comparison to the control cell population. By "attributable to" in this context is intended that the genomic alteration(s) is(are) directly or indirectly responsible for the increase in extracellular secretion. Thus, an improved cell population having one or more genomic alterations attributing to the increase in extracellular secretion will exhibit an increased level of secretion of a heterologous protein compared to the level of secretion from an otherwise genetically identical cell population expressing the same heterologous protein.

While not being bound to any particular theory or mechanism, the improved secretion may be the result of one or more genomic alterations in the P.fluorescens- derived cells affecting one or more genes involved in outer membrane vesicle (OMV) formation and/or function. OMVs are composed mainly of outer membrane proteins, lipopolysaccharide (LPS), outer membrane lipids and periplasmic proteins, and are produced during the normal growth cycle of cells. Formed after budding-off, or blebbing, from the outer membrane, spherical OMVs reach an average diameter of 20-250 nm (Keenan et al. (2000) FEMS Microbiol. Lett. 182, 259-264).

Gram-negative bacteria have the natural ability to release protein from the periplasm into the culture medium by production of OMVs (Horstman and Kuehn (2000) J. Biol. Chem. 275, 12489-1249). Thus, mutations in proteins linking the outer membrane to the peptidoglycan layer, or in the structural network between the inner and outer membranes of Gram-negative bacteria, can result in increased OMV release (Beveridge (1999) J. Bacteriol. 181, 4725-4733; Horstman and Kuehn (2000) J. Biol. Chem. 275, 12489-12496; Kato et al. (2002) Microb. Pathog. 32, 1-13; and, Wai et al. (1995) Microbiol. Immunol. 39, 451-456). In E. coli, a number of hyper- vesiculating mutants have been identified with mutations in the tol-pal gene cluster (Beveridge (1999) supra). The Tol-Pal system consists of seven proteins involved in the maintenance of outer membrane structure. Homologues of the tol-pal genes have also been found in many Gram-negative bacteria including Pseudomonas aeruginosa and Pseudomonas putida (Kato (2002) supra, and Li et al. (1998) J. Bacteriol. 180, 5478-5483).

Thus, in various embodiments of the present invention, the improved host cells described herein have one or more genomic alterations affecting the formation and/or blebbing of OMVs. The mutation may be in one or more genes homologous to members of the ToI-P al system in E. coli. For example, the mutation may be in one or more genes homologous to ToIC, ToIA, ToIQ, ToIR, ToIB, Pal, Orfl, or Orf2. Alternatively, or in addition, the genomic alterations may be in any gene encoding a protein involved in linking the outer membrane to the peptidoglycan layer, or encoding a protein in the structural network between the inner and outer membranes, each of which may lead to increased OMV formation. Various methods for purification and quantification of OMV production in various Gram-negative bacteria have been published (see, for example, Bernadac et al. (1998) Journal of Bacteriology 180(18):4872-4878, herein incorporated by reference in its entirety).

It is also contemplated that any other pathway or mechanism may be responsible for improving extracellular secretion of heterologous protein from the improved P. fluorescens-dcήvQd strains of the invention including, for example, improved expression or stability of the secreted protein, improved targeting of the protein to the extracellular space via mechanisms other than OMVs, and improved release into the extracellular space of proteins targeted to the periplasm.

The improved host cell populations described herein may be used for generating large quantities of a protein or polypeptide of interest. To further improve secretion into the extracellular medium of these proteins of interest, the host cell populations may be used in combination with cell lysis proteins to facilitate membrane permeabilization, for example, bacteriocin release protein, or BRP (see, for example, Rahman et al. (2005) Protein Expression and Purification 40:411-416) or killing protein (see, for example, Mahsunah et al. (2003) Appl Environ Microbiol 69:1237-1245). The cell populations may also be used with glycine and detergent supplementation to enhance release of periplasmic protein into the culture media (see, for example, Yang et al. (1998) Appl Environ Microbiol 64:2669-2874). Likewise, the rate of secretion may be increased by using a variety of other mechanisms that permeabilize the outer cell membrane, including: colicin (Miksch et al. (1997) Arch. Microbiol. 167: 143-150); growth rate (Shokri et al. (2002) App Miocrobiol Biotechnol 58:386-392); ToIIII overexpression (Wan and Baneyx (1998) Protein Expression Purif. 14: 13-22); bacteriocin release protein (Hsiung et al. (1989) Bio /Technology 7: 267-71); fusion partners (Jeong and Lee (2002) Appl. Environ. Microbio. 68: 4979-4985); or, recovery by osmotic shock (Taguchi et al. (1990) Biochimica Biophysica Acta 1049: 278-85).

Genome Modification

One or more host cell populations of the invention can be modified by any technique known in the art, for example by a technique wherein at least one gene affecting extracellular secretion (i.e., "target gene") is knocked out of the genome, or by mutating at least one target gene to reduce expression of the gene, by altering the promoter of at least one target gene to reduce expression of the target gene, and the like.

Targeted mutation

The genome of the host cell can be modified via a genetic targeting event, which can be by insertion or recombination, for example homologous recombination. Homologous recombination refers to the process of DNA recombination based on sequence homology. Homologous recombination permits site-specific modifications in endogenous genes and thus novel alterations can be engineered into a genome (see, for example Radding (1982) Ann. Rev. Genet. 16: 405; U.S. Pat. No. 4,888,274).

Various constructs can be prepared for homologous recombination at a target locus. Usually, the construct can include at least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous with the identified locus. Various considerations can be involved in determining the extent of homology of target gene sequences, such as, for example, the size of the target locus, availability of sequences, relative efficiency of double cross-over events at the target locus and the similarity of the target sequence with other sequences. The modified gene can include a sequence in which DNA substantially isogenic flanks the desired sequence modifications with a corresponding target sequence in the genome to be modified. The "modified gene" is the sequence being introduced into the host cell genome to increase (either directly or indirectly) the level of extracellular secretion of a heterologous protein from the host cell. The "target gene" is the sequence that is being replaced by the modified gene. The substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (except for the desired sequence modifications). The modified gene and the targeted gene can share stretches of DNA at least about 10, 20, 30, 50, 75, 150 or 500 base pairs that are 100% identical.

Nucleotide constructs can be designed to modify the endogenous, target gene product. The modified gene sequence can have one or more deletions, insertions, substitutions or combinations thereof designed to disrupt the function of the resultant gene product. In one embodiment, the alteration can be the insertion of a selectable marker gene fused in reading frame with the upstream sequence of the target gene.

The genome can also be modified using insertional inactivation. In this embodiment, the genome is modified by recombining a sequence in the gene that inhibits gene product formation. This insertion can either disrupt the gene by inserting a separate element, or remove an essential portion of the gene. In one embodiment, the insertional deletion also includes insertion of a gene coding for resistance to a particular stressor, such as an antibiotic, or for growth in a particular media, for example for production of an essential amino acid.

The genome can also be modified by use of transposons, which are genetic elements capable of inserting at sites in prokaryote genomes by mechanisms independant of homologous recombination. Transposons can include, for example,

Tn7, Tn5, or TnIO in E. coli, Tn554 in S. aureus, IS900 in M. paratuberculosis, IS492 from Pseudomonas atlantica, ISl 16 from Streptomyces and IS900 from M. paratuberculosis. Steps believed to be involved in transposition include cleavage of the end of the transposon to yield 3' OH; strand transfer, in which transposase brings together the 3 'OH exposed end of transposon and the identified sequence; and a single step transesterification reaction to yield a covalent linkage of the transposon to the identified DNA. The key reaction performed by transposase is generally thought to be nicking or strand exchange, the rest of the process is done by host enzymes.

In one embodiment, the expression or activity of the target gene is decreased by recombination with an inactive gene. The mutated version of the target gene may not encode a protein, or the protein encoded by the mutated gene may be rendered inactive, the activity may be modulated (either increased or decreased), or the mutant protein can have a different activity when compared to the native protein.

There are strategies to knock out genes in bacteria, which have been generally exemplified in E. coli. One route is to clone a gene-intemal DNA fragment into a vector containing an antibiotic resistance gene (e.g. ampicillin). Before cells are transformed via conjugative transfer, chemical transformation or electroporation (Puehler, et al. (1984) Advanced Molecular Genetics New York, Heidelberg, Berlin, Tokyo, Springer Verlag), an origin of replication, such as the vegetative plasmid replication (the oriV locus) is excised and the remaining DNA fragment is re-ligated and purified (Sambrook, et al. (2000) Molecular cloning: A laboratory manual, third edition Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press). Alternatively, antibiotic-resistant plasmids that have a DNA replication origin can be used. After transformation, the cells are plated onto e.g. LB agar plates containing the appropriate antibiotics (e.g. 200 micrograms/mL ampicillin). Colonies that grow on the plates containing the antibiotics presumably have undergone a single recombination event (Snyder, L., W. Champness, et al. (1997) Molecular Genetics of Bacteria Washington DC, ASM Press) that leads to the integration of the entire DNA fragment into the genome at the homologous locus. Further analysis of the antibiotic- resistant cells to verify that the desired gene knock-out has occurred at the desired locus is e.g. by diagnostic PCR (McPherson, M. J., P. Quirke, et al. (1991) PCR: A Practical Approach New York, Oxford University Press). Here, at least two PCR primers are designed: one that hybridizes outside the DNA region that was used for the construction of the gene knock-out; and one that hybridizes within the remaining plasmid backbone. Successful PCR amplification of the DNA fragment with the correct size followed by DNA sequence analysis will verify that the gene knock-out has occurred at the correct location in the bacterial chromosome. The phenotype of the newly constructed mutant strain can then be analyzed by, e.g., SDS polyacrylamide gel electrophoresis (Simpson, R. J. (2003) Proteins and Proteomics— A Laboratory Manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press).

An alternate route to generate a gene knock-out is by use of a temperature- sensitive replicon, such as the pSClOl replicon to facilitate gene replacement (Hamilton, et al. (1989) Journal of Bacteriology 171(9): 4617-22). The process proceeds by homologous recombination between a gene on a chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. After transformation of the plasmid into the appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44° C. Subsequent growth of these cointegrates at 30° C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene.

Random mutagenesis

Improved strains can also be developed through random mutagenesis and selection strategies. In this embodiment, a wildtype P. fluorescens cell population is transformed with an expression construct encoding a protein or polypeptide of interest. The population is cultured under suitable conditions and subsequently exposed to a mutagenic agent. The cells may be exposed to a chemical mutagen such as 5-bromo-deoxyuridine (5BU), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES), nitrosoguanidine (NTG, NG, MNNG), N- ethyl-N-nitrosourea (EΝU) or nitrous acid, or the cells may be irradiation using, for example, ultraviolet radiation or gamma radiation. The time and conditions of exposure will vary depending on the mutagen. One of skill in the art will understand appropriate techniques for optimizing such conditions.

Following exposure to the mutagen, the cells can be grown as independent colonies (e.g., by streaking the cells onto a solid or semi-solid media such as agar), picked, and inoculated into fresh media. The individual colonies can be cultured under suitable conditions for expression of the heterologous protein or polypeptide of interest, and cell-free extracts obtained from the culture to identify strains having improved extracellular secretion.

For the purposes of the present invention, the term "increased" or "improved" in the context of extracellular secretion is relative to the level of protein or polypeptide that is secreted into the extracellular space when the protein or polypeptide of interest is expressed in one or more control cell populations. In one embodiment, the improved host cell population secretes into the extracellular media at least 0.1 mg of the heterologous protein of interest per ml of cell culture media when expressed at an optical cell density of at least 40 mg/ml, when grown (i.e. within a temperature range of about 4°C to about 55°C, including about 10⁰C, about 15 °C, about 20⁰C, about 25⁰C, about 30⁰C, about 35⁰C, about 40⁰C, about 45⁰C, and about 5O⁰C) in a mineral salts medium. In another embodiment, the improved strain secretes 0.1 to 10 mg/ml protein, or at least about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9 or at least about 1.0 mg/ml protein. In one embodiment, the total protein or polypeptide of interest produced by the improved host cell population of the invention is at least 1.0 mg/ml, at least about 2 mg/ml, at least about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, at least about 25 mg/ml, or greater. In some embodiments, the amount of heterologous protein of interest that is secreted to the extracellular space is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more of the total heterologous protein of interest produced by the cell population. In another embodiment, the improved host cell population secretes at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 2-fold, at least about 3-fold, at least about 4-fold or more of the protein of interest into the extracellular space when compared to a control cell population. In one embodiment, the improvement in secretion is determined based on the amount or activity of the protein of interest. While the protein of interest may be any protein or polypeptide that is detectable in the culture media, it will be useful to utilize a reporter protein for developing and screening mutant cell populations. By "reporter protein" is meant a protein that, when secreted in the media, facilitates detection and quantification of the protein in the media. The reporter protein can be detected in the extracellular media based on the presence or activity, or both, of the reporter protein. The reporter protein can be firefly luciferase, green fluorescent protein (GFP), or any other fluorescence molecule, as well as alkaline phosphatase, beta-galactosidase, and the chloramphenicol and acetyltransferase gene (CAT). Assays for expression produced in conjunction with each of these reporter gene elements are well known to those skilled in the art.

The reporter gene can encode a detectable protein or an indirectly detectable protein, or the reporter gene can be a survival gene. In a preferred embodiment, the reporter protein is a detectable protein. A "detectable protein" or "detection protein" (encoded by a detectable or detection gene) is a protein that can be used as a direct label; that is, the protein is detectable (and preferably, a cell culture media comprising the detectable protein is detectable) without further manipulation. Thus, in this embodiment, the protein product of the reporter gene itself can serve to assess the level of heterologous protein secretion. In this embodiment, suitable detectable genes include those encoding auto fluorescent proteins. Preferably, the reporter protein is suitable for quantitative assessment of protein secretion but qualitative indicators are also encompassed.

In another embodiment, the amount of secretion is determined based on the quantity of the heterologous protein of interest detectable in the cell-free media.

Thus, the method may also include the step of purifying the protein or polypeptide of interest from the extracellular media. The heterologous protein or polypeptide can be expressed in a manner in which it is linked to a tag protein and the "tagged" protein can be purified from the cell or extracellular media. To measure the yield and/or activity of the secreted protein of interest, it may be desirable to isolate the protein from one or more strains in the array. The isolation may be a crude, semi-crude, or pure isolation, depending on the requirements of the assay used to make the appropriate measurements.

If desired, the proteins produced using one or more strains in the array of this invention may be isolated and purified to substantial purity by standard techniques well known in the art, including, but not limited to, ammonium sulfate or ethanol precipitation, centrifugation, filtration, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, nickel chromatography, hydroxylapatite chromatography, reverse phase chromatography, lectin chromatography, preparative electrophoresis, detergent solubilization, selective precipitation with such substances as column chromatography, immunopurification methods, and others. For example, proteins having established molecular adhesion properties can be reversibly fused with a ligand. With the appropriate ligand, the protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. In addition, protein can be purified using immunoaffinity columns or Ni-NTA columns. General techniques are further described in, for example, R. Scopes, Protein Purification: Principles and Practice, Springer- Verlag: N.Y. (1982); Deutscher, Guide to Protein Purification, Academic Press (1990); U.S. Pat. No. 4,511 ,503; S. Roe, Protein Purification

Techniques: A Practical Approach (Practical Approach Series), Oxford Press (2001); D. Bollag, et al, Protein Methods, Wiley-Lisa, Inc. (1996); AK Patra et al, Protein Expr Purif, 18(2): p/182-92 (2000); and R. Mukhija, et al, Gene 165(2): p. 303-6 (1995). See also, for example, Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification," Methods in Enzymology vol. 182, and other volumes in this series; Coligan, et al. (1996 and periodic Supplements) Current Protocols in Protein Science Wiley/Greene, NY; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N. J., or Bio-Rad, Richmond, Calif. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See also, for example., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY; and Crowe, et al. (1992) QIAexpress: The High Level Expression & Protein Purification System QIAGEN, Inc., Chatsworth, Calif.

Detection of the expressed and secreted protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation. In other embodiments, the protein or polypeptide of interest is secreted by the improved host cell population in an active form, and the level of activity can be assayed in the media, or in a purified or semi-purified sample of the protein. The term "active" means the presence of biological activity, wherein the biological activity is comparable or substantially corresponds to the biological activity of a corresponding native protein or polypeptide. In the context of proteins this typically means that a polynucleotide or polypeptide comprises a biological function or effect that has at least about 20%, about 50%, preferably at least about 60-80%, and most preferably at least about 90-95% activity compared to the corresponding native protein or polypeptide using standard parameters. However, in some embodiments, it may be desirable to produce a polypeptide that has altered or improved activity compared to the native protein (e.g, one that has altered or improved immunoreactivity, substrate specificity, etc). An altered or improved polypeptide may result from a particular conformation created by the improved host cell population of the invention. The determination of protein or polypeptide activity can be performed utilizing corresponding standard, targeted comparative biological assays for particular proteins or polypeptides which can be used to assess biological activity.

Active proteins can have a specific activity of at least about 20%, at least about 30%, at least about 40%, about 50%, about 60%, at least about 70%, about 80%, about 90%, or at least about 95% that of the native protein or polypeptide from which the sequence is derived. Further, the substrate specificity (k_cat/K_m) is optionally substantially similar to the native protein or polypeptide. Typically, k_cat/K_m will be at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, at least about 90%, at least about 95%, or greater. Methods of assaying and quantifying measures of protein and polypeptide activity and substrate specificity (k_cat/K_m), are well known to those of skill in the art.

The activity of the heterologously-expressed protein or polypeptide of interest can be compared with a previously established native protein or polypeptide standard activity. Alternatively, the activity of the protein or polypeptide of interest can be determined in a simultaneous, or substantially simultaneous, comparative assay with the native protein or polypeptide. For example, in vitro assays can be used to determine any detectable interaction between a protein or polypeptide of interest and a target, e.g. between an expressed enzyme and substrate, between expressed hormone and hormone receptor, between expressed antibody and antigen, etc. Such detection can include the measurement of calorimetric changes, proliferation changes, cell death, cell repelling, changes in radioactivity, changes in solubility, changes in molecular weight as measured by gel electrophoresis and/or gel exclusion methods, phosphorylation abilities, antibody specificity assays such as ELISA assays, etc. In addition, in vivo assays include, but are not limited to, assays to detect physiological effects of the heterologously expressed protein or polypeptide in comparison to physiological effects of the native protein or polypeptide, e.g. weight gain, change in electrolyte balance, change in blood clotting time, changes in clot dissolution and the induction of antigenic response. Generally, any in vitro or in vivo assay can be used to determine the active nature of the protein or polypeptide of interest that allows for a comparative analysis to the native protein or polypeptide so long as such activity is assayable. Alternatively, the proteins or polypeptides produced in the improved host cell population of the present invention can be assayed for the ability to stimulate or inhibit interaction between the protein or polypeptide and a molecule that normally interacts with the protein or polypeptide, e.g. a substrate or a component of a signal pathway with which the native protein normally interacts. Such assays can typically include the steps of combining the protein with a substrate molecule under conditions that allow the protein or polypeptide to interact with the target molecule, and detect the biochemical consequence of the interaction with the protein and the target molecule. Assays that can be utilized to determine protein or polypeptide activity are described, for example, in Ralph, P. J., et al. (1984) J. Immunol. 132:1858 or Saiki et al (198I) J. Immunol. 127:1044, Steward, W. E. II (1980) The Interferon Systems. Springer-Verlag, Vienna and New York, Broxmeyer, H. E., et al. (1982) Blood 60:595, Molecular Cloning: A Laboratory Manua" , 2d ed., Cold Spring Harbor

Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and Methods in Enzymology: Guide to Molecular Cloning Techniques, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987, A K Patra et al, Protein Expr Purif, 18(2): p/182-92 (2000), Kodama et al., J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115 (1995); (Vale et al., Cell 42:39-50 (1985). Activity can be compared between samples of heterologously expressed protein derived from one or more control host cell populations, or can be compared to the activity of a native protein, or both. Activity measurements can be performed on isolated protein. The level of extracellular secretion may also be monitored directly in the culture by fluorescence or spectroscopic measurements on, for example, a conventional microscope, luminometer, or plate reader. Where the protein of interest is an enzyme whose substrate is known, the substrate can be added to the culture media wherein a fluorescent signal is emitted when the substrate is converted by the enzyme into a product.

As is known in the art, there are a variety of autofluorescent proteins known; these generally are based on the green fluorescent protein (GFP) from Aequorea and variants thereof; including, but not limited to, GFP, (Chalfie, et al. (1994) Science 263(5148):802-805); enhanced GFP (EGFP; Clontech-Genbank Accession Number U55762)), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc., Montreal, Canada); Stauber (1998) Biotechniques 24(3):462-471; Heim and Tsien(1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP; Clontech Laboratories, Inc., Palo Alto, CA) and red fluorescent protein. In addition, there are recent reports of autofluorescent proteins from Renilla and Ptilosarcus species. See WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No.

5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558; all of which are expressly incorporated herein by reference.

Expression vectors

A heterologous protein of interest can be produced in one or more of the improved host cells disclosed herein by introducing into the host cell an expression vector encoding the heterologous protein of interest. In one embodiment, the vector comprises a polynucleotide sequence encoding the protein of interest operably linked to a promoter capable of functioning in the chosen host cell, as well as all other required transcription and translation regulatory elements. The term "operably linked" refers to any configuration in which the transcriptional and any translational regulatory elements are covalently attached to the encoding sequence in such disposition(s), relative to the coding sequence, that in and by action of the host cell, the regulatory elements can direct the expression of the coding sequence. The heterologous protein of interest can be expressed from polynucleotides in which the heterologous polypeptide coding sequence is operably linked to transcription and translation regulatory elements to form a functional gene from which the host cell can express the protein or polypeptide. The coding sequence for the protein or polypeptide of interest can be a native coding sequence for the polypeptide, if available, but will more preferably be a coding sequence that has been selected, improved, or optimized for use in an expressible form in the strains of the invention: for example, by optimizing the gene to reflect the codon use bias of a Pseudomonas species such as P. fluorescens or other suitable organism. For gene optimization, one or more rare codons may be removed to avoid ribosomal stalling and minimize amino acid misincorporation. One or more gene-internal ribosome binding sites may also be eliminated to avoid truncated protein products. Long stretches of C and G nucleotides may be removed to avoid RNA polymerase slippage that could result in frame-shifts. Strong gene-internal stem-loop structures, especially the ones covering the ribosome binding site, may also be eliminated. The gene(s) are constructed within or inserted into one or more vector(s), which can then be transformed into the expression host cell.

Other regulatory elements may be included in a vector (also termed "expression construct"). The vector will typically comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Additional elements include, but are not limited to, for example, transcriptional enhancer sequences, translational enhancer sequences, other promoters, activators, translational start and stop signals, transcription terminators, cistronic regulators, polycistronic regulators, or tag sequences, such as nucleotide sequence "tags" and "tag" polypeptide coding sequences, which facilitates identification, separation, purification, and/or isolation of an expressed polypeptide.

In another embodiment, the expression vector further comprises a tag sequence adjacent to the coding sequence for the protein or polypeptide of interest. In one embodiment, this tag sequence allows for purification of the protein. The tag sequence can be an affinity tag, such as a hexa-histidine affinity tag. In another embodiment, the affinity tag can be a glutathione-S-transferase molecule. The tag can also be a fluorescent molecule, such as yellow fluorescent protein (YFP) or green fluorescent protein (GFP), or analogs of such fluorescent proteins. The tag can also be a portion of an antibody molecule, or a known antigen or ligand for a known binding partner useful for purification.

A protein-encoding gene according to the present invention can include, in addition to the protein coding sequence, the following regulatory elements operably linked thereto: a promoter, a ribosome binding site (RBS), a transcription terminator, translational start and stop signals. Useful RBSs can be obtained from any of the species useful as host cells in expression systems according to the present invention, preferably from the selected host cell. Many specific and a variety of consensus RBSs are known, e.g., those described in and referenced by D. Frishman et al, Gene 234(2):257-65 (8 JuI. 1999); and B. E. Suzek et al, Bioinformatics 17(12): 1123-30 (December 2001). In addition, either native or synthetic RBSs may be used, e.g., those described in: EP 0207459 (synthetic RBSs); O. Ikehata et al, Eur. J. Biochem. 181(3):563-70 (1989) (native RBS sequence of AAGGAAG). Further examples of methods, vectors, and translation and transcription elements, and other elements useful in the present invention are described in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy et al; U.S. Pat. No. 5,281,532 to Rammler et al; U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al; U.S. Pat. No. 4,755,465 to Gray et al; and U.S. Pat. No. 5,169,760 to Wilcox.

Transcription of the DNA encoding the heterologous protein of interest is increased by inserting an enhancer sequence into the vector or plasmid. Typical enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp in size that act on the promoter to increase its transcription. Examples include various Pseudomonas enhancers.

Generally, the heterologous expression vectors will include origins of replication and selectable markers permitting transformation of the host cell and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding the enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others. Where signal sequences are used, the heterologous coding sequence is assembled in appropriate phase with translation initiation and termination sequences, and the signal sequence capable of directing compartmental accumulation or secretion of the translated protein. Optionally the heterologous sequence can encode a fusion enzyme including an N-terminal identification polypeptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed heterologous product. The fusion polypeptide can also comprise one or more target proteins or inhibitors or enhances thereof, as discussed supra.

In other embodiments, the protein when produced also includes an additional targeting sequence, for example a sequence that targets the protein to the extracellular medium. In one embodiment, the additional targeting sequence is operably linked to the carboxy-terminus of the protein. A number of secretion signals have been described for use in expressing recombinant polypeptides or proteins. See, for example, U.S. Pat. No. 5,914,254; U.S. Pat. No. 4,963,495; European Patent No. 0 177 343; U.S. Pat. No. 5,082,783; PCT Publication No. WO 89/10971; U.S. Pat. No. 6,156,552; U.S. Pat. Nos. 6,495,357; 6,509,181; 6,524,827; 6,528,298; 6,558,939; 6,608,018; 6,617,143; U.S. Pat. Nos. 5,595,898; 5,698,435; and 6,204,023; U.S. Pat. No. 6,258,560; PCT Publication Nos. WO 01/21662, WO 02/068660 and U.S. Application Publication 2003/0044906; U.S. Pat. No. 5,641,671; and European Patent No. EP 0 121 352. Expression constructs may further comprise secretion leaders, outer membrane fusion partners (e.g., OmpF; see Jeong and Lee (2002) Applied and Environmental Microbiology 68:4979-4985), or hemolysin secretion signal (HIyAs; see, for example, Li et al. (2002) Protein Expression and Purification 25(3):437-447; Jeong et al. (2002) Applied and Environmental Microbiology 68:4979-4985; Mergulhao et al. (2005) Biotechnology Advances 23:177-202; and, Rahman et al. (2005) Protein Expression and Purification 40:411-416, each of which is herein incorporated by reference in its entirety). The polypeptide of interest may also be coexpressed with genes encoding proteins capable of improving outer membrane vesicle formation, such as those involved in or capable of disrupting the Tol-Pal system of Escherichia coli, or those required to maintain outer membrane integrity, as described elsewhere herein.

Vectors are known in the art for expressing heterologous proteins in host cells, and any of these may be used for expressing the genes according to the present invention. Such vectors include, e.g., plasmids, cosmids, and phage expression vectors. Examples of useful plasmid vectors include, but are not limited to, the expression plasmids pBBRIMCS, pDSK519, pKT240, pML122, pPSIO, RK2, RK6, pRO1600, and RSFlOlO. Other examples of such useful vectors include those described by, e.g.: N. Hayase, in Appl. Envir. Microbiol. 60(9):3336-42 (September 1994); A. A. Lushnikov et al., in Basic Life Sci. 30:657-62 (1985); S. Graupner & W. Wackemagel, in Biomolec. Eng. 17(1): 11-16. (October 2000); H. P. Schweizer, in Curr. Opin. Biotech. 12(5):439-45 (October 2001); M. Bagdasarian & K. N. Timmis, in Curr. Topics Microbiol. Immunol. 96:47-67 (1982); T. Ishii et al., in FEMS Microbiol. Lett. 116(3):307-13 (Mar. 1, 1994); I. N. Olekhnovich & Y. K. Fomichev, in Gene 140(l):63-65 (Mar. 11, 1994); M. Tsuda & T. Nakazawa, in Gene 136(1-

2):257-62 (Dec. 22, 1993); C. Nieto et al, in Gene 87(l):145-49 (Mar. 1, 1990); J. D. Jones & N. Gutterson, in Gene 61(3):299-306 (1987); M. Bagdasarian et al, in Gene 16(l-3):237-47 (December 1981); H. P. Schweizer et al, in Genet. Eng. (NY) 23:69- 81 (2001); P. Mukhopadhyay et al , in J. Bact. 172(l):477-80 (January 1990); D. O. Wood et al, in J. Bact. 145(3): 1448-51 (March 1981); and R. Holtwick et al, in Microbiology 147(Pt 2):337-44 (February 2001).

Further examples of expression vectors that can be useful in a host cell of the invention include those listed in Table 1 as derived from the indicated replicons.

The expression plasmid, RSFlOlO, is described, e.g., by F. Heffron et ah, in Proc. Nat'l Acad. Sci. USA 72(9):3623-27 (September 1975), and by K. Nagahari & K. Sakaguchi, in J. Bact. 133(3): 1527-29 (March 1978). Plasmid RSFlOlO and derivatives thereof are particularly useful vectors in the present invention. Exemplary useful derivatives of RSFlOlO, which are known in the art, include, e.g., pKT212, pKT214, pKT231 and related plasmids, and pMYC1050 and related plasmids (see, e.g., U.S. Pat. Nos. 5,527,883 and 5,840,554 to Thompson et al.), such as, e.g., pMYC1803. Plasmid pMYC1803 is derived from the RSF1010-based plasmid pTJS260 (see U.S. Pat. No. 5,169,760 to Wilcox), which carries a regulated tetracycline resistance marker and the replication and mobilization loci from the RSFlOlO plasmid. Other exemplary useful vectors include those described in U.S. Pat. No. 4,680,264 to Puhler et al.

In one embodiment, an expression plasmid is used as the expression vector. In another embodiment, RSF 1010 or a derivative thereof is used as the expression vector. In still another embodiment, pMYC1050 or a derivative thereof, or pMYC4803 or a derivative thereof, is used as the expression vector.

The plasmid can be maintained in the host cell by inclusion of a selection marker gene in the plasmid. This may be an antibiotic resistance gene(s), where the corresponding antibiotic(s) is added to the fermentation medium, or any other type of selection marker gene known in the art, e.g., a prototrophy-restoring gene where the plasmid is used in a host cell that is auxotrophic for the corresponding trait, e.g., a biocatalytic trait such as an amino acid biosynthesis or a nucleotide biosynthesis trait, or a carbon source utilization trait. The promoters used in accordance with the present invention may be constitutive promoters or regulated promoters. Common examples of useful regulated promoters include those of the family derived from the lac promoter (i.e. the lacZ promoter), especially the tac and trc promoters described in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptacl6, Ptacl7, PtacII, PlacUV5, and the T71ac promoter. In one embodiment, the promoter is not derived from the host cell organism. In certain embodiments, the promoter is derived from an E. coli organism.

Common examples of non-lac-type promoters useful in expression systems according to the present invention include, e.g., those listed in Table 2.

See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Manual of Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washington, D. C); H. Schweizer (2001) Current Opinion in Biotechnology, 12:439-445; and R. Slater & R. Williams (2000 Molecular Biology and

Biotechnology (J. Walker & R. Rapley, eds.) pp. 125-54 (The Royal Society of Chemistry, Cambridge, UK)). A promoter having the nucleotide sequence of a promoter native to the selected bacterial host cell may also be used to control expression of the transgene encoding the target polypeptide, e.g, a Pseudomonas anthranilate or benzoate operon promoter (Pant, Pben). Tandem promoters may also be used in which more than one promoter is covalently attached to another, whether the same or different in sequence, e.g., a Pant-Pben tandem promoter (interpromoter hybrid) or a Plac-Plac tandem promoter, or whether derived from the same or different organisms. Regulated promoters utilize promoter regulatory proteins in order to control transcription of the gene of which the promoter is a part. Where a regulated promoter is used herein, a corresponding promoter regulatory protein will also be part of an expression system according to the present invention. Examples of promoter regulatory proteins include: activator proteins, e.g., E. coli catabolite activator protein, MaIT protein; AraC family transcriptional activators; repressor proteins, e.g., E. coli Lad proteins; and dual-function regulatory proteins, e.g., E. coli NagC protein. Many regulated-promoter/promoter-regulatory-protein pairs are known in the art. In one embodiment, the expression construct for the target protein(s) and the heterologous protein of interest are under the control of the same regulatory element. Promoter regulatory proteins interact with an effector compound, i.e. a compound that reversibly or irreversibly associates with the regulatory protein so as to enable the protein to either release or bind to at least one DNA transcription regulatory region of the gene that is under the control of the promoter, thereby permitting or blocking the action of a transcriptase enzyme in initiating transcription of the gene. Effector compounds are classified as either inducers or co-repressors, and these compounds include native effector compounds and gratuitous inducer compounds. Many regulated-promoter/promoter-regulatory-protein/effector- compound trios are known in the art. Although an effector compound can be used throughout the cell culture or fermentation, in a preferred embodiment in which a regulated promoter is used, after growth of a desired quantity or density of host cell biomass, an appropriate effector compound is added to the culture to directly or indirectly result in expression of the desired gene(s) encoding the protein or polypeptide of interest. By way of example, where a lac family promoter is utilized, a lad gene can also be present in the system. The lad gene, which is (normally) a constitutively expressed gene, encodes the Lac repressor protein (LacD protein) which binds to the lac operator of these promoters. Thus, where a lac family promoter is utilized, the lad gene can also be included and expressed in the expression system. In the case of the lac promoter family members, e.g., the tac promoter, the effector compound is an inducer, preferably a gratuitous inducer such as IPTG (isopropyl-D- 1 - thiogalactopyranoside, also called "isopropylthiogalactoside").

Expression Systems In one embodiment, the expression vector further comprises a nucleotide sequence encoding a secretion signal sequence polypeptide operably linked to the nucleotide sequence encoding the protein or polypeptide of interest. In some embodiments, no modifications are made between the signal sequence and the protein or polypeptide of interest. However, in certain embodiments, additional cleavage signals are incorporated to promote proper processing of the amino terminal of the polypeptide.

The vector can have any of the characteristics described above. In one embodiment, the vector comprising the coding sequence for the protein or polypeptide of interest further comprises a signal sequence, e.g., a secretion signal sequence. Therefore, in one embodiment, this isolated polypeptide is a fusion protein of the secretion signal and a protein or polypeptide of interest.

The CHAMPION™ pET expression system provides a high level of protein production. Expression is induced from the strong T71ac promoter. This system takes advantage of the high activity and specificity of the bacteriophage T7 RNA polymerase for high level transcription of the gene of interest. The lac operator located in the promoter region provides tighter regulation than traditional T7-based vectors, improving plasmid stability and cell viability (Studier and Moffatt (1986) J Molecular Biology 189(1): 113-30; Rosenberg, et al. (1987) Gene 56(1): 125-35). The T7 expression system uses the T7 promoter and T7 RNA polymerase (T7 RNAP) for high-level transcription of the gene of interest. High-level expression is achieved in T7 expression systems because the T7 RNAP is more processive than native E. coli RNAP and is dedicated to the transcription of the gene of interest. Expression of the identified gene is induced by providing a source of T7 RNAP in the host cell. This is accomplished by using a BL21 E. coli host containing a chromosomal copy of the T7 RNAP gene. The T7 RNAP gene is under the control of the lacUV5 promoter which can be induced by IPTG. T7 RNAP is expressed upon induction and transcribes the gene of interest.

The pBAD expression system allows tightly controlled, titratable expression of protein or polypeptide of interest through the presence of specific carbon sources such as glucose, glycerol and arabinose (Guzman, et al. (1995) J Bacteriology 177(14): 4121-30). The pBAD vectors are uniquely designed to give precise control over expression levels. Heterologous gene expression from the pBAD vectors is initiated at the araBAD promoter. The promoter is both positively and negatively regulated by the product of the araC gene. AraC is a transcriptional regulator that forms a complex with L-arabinose. In the absence of L-arabinose, the AraC dimer blocks transcription. For maximum transcriptional activation two events are required: (i) L-arabinose binds to AraC allowing transcription to begin, and, (ii) The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to the correct location of the promoter region.

The trc expression system allows high-level, regulated expression in E. coli from the trc promoter. The trc expression vectors have been optimized for expression of eukaryotic genes in E. coli. The trc promoter is a strong hybrid promoter derived from the tryptophane (trp) and lactose (lac) promoters. It is regulated by the lacO operator and the product of the lacIQ gene (Brosius, J. (1984) Gene 27(2): 161-72). Transformation of the host cells with the vector(s) disclosed herein may be performed using any transformation methodology known in the art, and the bacterial host cells may be transformed as intact cells or as protoplasts (i.e. including cytoplasts). Exemplary transformation methodologies include poration methodologies, e.g., electroporation, protoplast fusion, bacterial conjugation, and divalent cation treatment, e.g., calcium chloride treatment or CaCl/Mg2+ treatment, or other well known methods in the art. See, e.g., Morrison, J. Bact. , 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology, 101 :347-362 (Wu et al, eds, 1983), Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et ah, eds., 1994)).

Cell growth conditions

The cell growth conditions for the host cells described herein include that which facilitates expression of the protein of interest in the improved P. fluorescens cell population described herein, and/or that which facilitates fermentation of the expressed protein of interest. As used herein, the term "fermentation" includes both embodiments in which literal fermentation is employed and embodiments in which other, non-fermentative culture modes are employed. Growth, maintenance, and/or fermentation of the populations of improved host cells described herein may be performed at any scale. In one embodiment, the fermentation medium may be selected from among rich media, minimal media, and mineral salts media. In another embodiment either a minimal medium or a mineral salts medium is selected. In still another embodiment, a minimal medium is selected. In yet another embodiment, a mineral salts medium is selected.

Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol. Examples of mineral salts media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), Davis and Mingioli medium (see, BD Davis & ES Mingioli (1950) in J. Bad. 60:17-28). The mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc. No organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium. Instead, an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia. A preferred mineral salts medium will contain glucose as the carbon source. In comparison to mineral salts media, minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels.

In the present invention, growth, culturing, and/or fermentation of the transformed host cells is performed within a temperature range permitting survival of the host cells, preferably a temperature within the range of about 4°C to about 55⁰C, inclusive, preferably not more than about 34⁰C. Thus, e.g., the terms "growth" (and "grow," "growing"), "culturing" (and "culture"), and "fermentation" (and "ferment," "fermenting"), as used herein in regard to the host cells of the present invention, inherently means "growth," "culturing," and "fermentation," within a temperature range of about 4°C to about 55⁰C, inclusive, preferably not more than about 34°C. In addition, "growth" is used to indicate both biological states of active cell division and/or enlargement, as well as biological states in which a non-dividing and/or non- enlarging cell is being metabolically sustained, the latter use of the term "growth" being synonymous with the term "maintenance."

The host cells of the invention should be grown and maintained at a suitable temperature for normal growth of that cell type. Such normal growth temperatures may be readily selected based on the known growth requirements of the host cell. Preferably, during the establishment of the culture and particularly during course of the screening, the cell culture is incubated in a controlled CO₂/N₂ humidity suitable for growth of the selected cells before and after transformation with the heterologous protein or polypeptide of interest. The humidity of the incubation is controlled to minimize evaporation from the culture vessel, and permit the use of smaller volumes. Alternatively, or in addition to controlling humidity, the vessels may be covered with lids in order to minimize evaporation. Selection of the incubation temperature depends primarily upon the identity of the host cells utilized. Selection of the percent humidity to control evaporation is based upon the selected volume of the vessel and concentration and volume of the cell culture in the vessel, as well as upon the incubation temperature. Thus, the humidity may vary from about 10% to about 80%. It should be understood that selection of a suitable conditions is well within the skill of the art.

Proteins of interest The methods and compositions of the present invention are useful for the expression and extracellular secretion of high levels of a properly processed protein or polypeptide of interest. The improved host cell populations described herein are useful for production of a protein or polypeptide of interest of any species and of any size. However, in certain embodiments, the protein or polypeptide of interest is a therapeutically useful protein or polypeptide. In some embodiments, the protein can be a mammalian protein, for example a human protein, and can be, for example, a growth factor, a cytokine, a chemokine or a blood protein. The protein or polypeptide of interest can be processed in a similar manner to the native protein or polypeptide. In certain embodiments, the protein or polypeptide of interest is less than 100 kD, less than 50 kD, or less than 30 kD in size. In certain embodiments, the protein or polypeptide of interest is a polypeptide of at least about 5, 10, 15, 20, 30, 40, 50 or 100 or more amino acids.

The gene(s) that result are constructed within or are inserted into one or more vectors, and then transformed into each of the improved host cell populations. Nucleic acid or a polynucleotide said to be provided in an "expressible form" means nucleic acid or a polynucleotide that contains at least one gene that can be expressed by the one or more of the host cell populations of the invention.

Extensive sequence information required for molecular genetics and genetic engineering techniques is widely publicly available. Access to complete nucleotide sequences of mammalian, as well as human, genes, cDNA sequences, amino acid sequences and genomes can be obtained from GenBank at the website www.ncbi.nlm.nih.gov/Entrez. Additional information can also be obtained from GeneCards, an electronic encyclopedia integrating information about genes and their products and biomedical applications from the Weizmann Institute of Science Genome and Bioinformatics (bioinformatics.weizmann.ac.il/cards), nucleotide sequence information can be also obtained from the EMBL Nucleotide Sequence Database (www.ebi.ac.uk/embl/) or the DNA Databank or Japan (DDBJ, www.ddbi.nig.ac.ii/; additional sites for information on amino acid sequences include Georgetown's protein information resource website (www-nbrf.Reorgetown.edu/pirl) and Swiss-Prot (au.expasy.org/sprot/sprot-top.html).

Examples of proteins that can be expressed in this invention include molecules such as, e.g., renin, a growth hormone, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; α- 1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial naturietic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue- type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated polypeptide; a microbial protein, such as beta-lactamase; Dnase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT -4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin-1 (CT-I); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-β, including TGF-β l, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and - gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-I to IL-IO; anti-HER-2 antibody; superoxide dismutase; T- cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; antibodies; and fragments of any of the above-listed polypeptides. In certain embodiments, the protein or polypeptide can be selected from IL-I, IL-Ia, IL-Ib, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12, IL- 12elasti, IL-13, IL-15, IL-16, IL-18, IL-18BPa, IL-23, IL-24, VIP, erythropoietin, GM-CSF, G-CSF, M-CSF, platelet derived growth factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growth factor (FGF; e.g., α-FGF (FGF-I), β-FGF (FGF-2), FGF-3, FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors (e.g., IGF-I, IGF-2); tumor necrosis factors (e.g., TNF, Lymphotoxin), nerve growth factors (e.g., NGF), vascular endothelial growth factor (VEGF); interferons (e.g., IFN-α, IFN-β, IFN-γ); leukemia inhibitory factor (LIF); ciliary neurotrophic factor (CNTF); oncostatin M; stem cell factor (SCF); transforming growth factors (e.g., TGF-α, TGF- βl, TGF-β2, TGF-β3); TNF superfamily (e.g., LIGHT/TNFSF14, STALL- 1/TNFSF13B (BLy5, BAFF, THANK), TNFalpha/TNFSF2 and TWEAK/TNFSF12); or chemokines (BCA- 1/BLC-l, BRAK/Kec, CXCLl 6, CXCR3, ENA-78/LIX, Eotaxin-1, Eotaxin-2/MPIF-2, Exodus-2/SLC, Fractalkine/Neurotactin, GROalpha/MGSA, HCC-I, I-TAC, Lymphotactin/ATAC/SCM, MCP-1/MCAF, MCP-3, MCP-4, MDC/STCP-l/ABCD-1, MIP-I . quadrature., MIP-I . quadrature., MIP-2.quadrature./GRO.quadrature., MIP-3.quadrature./Exodus/LARC, MIP- 3/Exodus-3/ELC, MIP-4/PARC/DC-CKl, PF-4, RANTES, SDFl, TARC, TECK, microbial toxins, ADP ribosylating toxins, microbial or viral antigens). In one embodiment of the present invention, the protein of interest can be a multi-subunit protein or polypeptide. Multisubunit proteins that can be expressed include homomeric and heteromeric proteins. The multisubunit proteins may include two or more subunits that may be the same or different. For example, the protein may be a homomeric protein comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subunits. The protein also may be a heteromeric protein including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more subunits. Exemplary multisubunit proteins include: receptors including ion channel receptors; extracellular matrix proteins including chondroitin; collagen; immunomodulators including MHC proteins, full chain antibodies, and antibody fragments; enzymes including RNA polymerases, and DNA polymerases; and membrane proteins.

In another embodiment, the protein of interest can be a blood protein. The blood proteins expressed in this embodiment include but are not limited to carrier proteins, such as albumin, including human and bovine albumin, transferrin, recombinant transferrin half-molecules, haptoglobin, fibrinogen and other coagulation factors, complement components, immunoglobulins, enzyme inhibitors, precursors of substances such as angiotensin and bradykinin, insulin, endothelin, and globulin, including alpha, beta, and gamma-globulin, and other types of proteins, polypeptides, and fragments thereof found primarily in the blood of mammals. The amino acid sequences for numerous blood proteins have been reported (see, S. S. Baldwin (1993) Comp. Biochem Physiol. 106b:203-218), including the amino acid sequence for human serum albumin (Lawn, L. M., et al. (1981) Nucleic Acids Research, 9:6103- 6114.) and human serum transferrin (Yang, F. et al. (1984) Proc. Natl. Acad. Sci. USA 81 :2752-2756).

In another embodiment, the protein of interest can be an enzyme or co-factor. The enzymes and co-factors expressed in this embodiment include but are not limited to aldolases, amine oxidases, amino acid oxidases, aspartases, B 12 dependent enzymes, carboxypeptidases, carboxyesterases, carboxylases, chemotrypsin, CoA requiring enzymes, cyanohydrin synthetases, cystathione synthases, decarboxylases, dehydrogenases, alcohol dehydrogenases, dehydratases, diaphorases, dioxygenases, enoate reductases, epoxide hydrases, fumerases, galactose oxidases, glucose isomerases, glucose oxidases, glycosyltrasferases, methyltransferases, nitrile hydrases, nucleoside phosphorylases, oxidoreductases, oxynitilases, peptidases, glycosyltrasferases, peroxidases, enzymes fused to a therapeutically active polypeptide, tissue plasminogen activator; urokinase, reptilase, streptokinase; catalase, superoxide dismutase; Dnase, amino acid hydrolases (e.g., asparaginase, amidohydrolases); carboxypeptidases; proteases, trypsin, pepsin, chymotrypsin, papain, bromelain, collagenase; neuramimidase; lactase, maltase, sucrase, and arabinofuranosidases.

In another embodiment, the protein of interest can be a single chain, Fab fragment and/or full chain antibody or fragments or portions thereof. A single-chain antibody can include the antigen-binding regions of antibodies on a single stably- folded polypeptide chain. Fab fragments can be a piece of a particular antibody. The Fab fragment can contain the antigen binding site. The Fab fragment can contain 2 chains: a light chain and a heavy chain fragment. These fragments can be linked via a linker or a disulfide bond.

In other embodiments, the protein of interest is a protein that is active at a temperature from about 20 to about 42⁰C. In one embodiment, the protein is active at physiological temperatures and is inactivated when heated to high or extreme temperatures, such as temperatures over 65⁰C.

In one embodiment, the protein of interest is a protein that is active at a temperature from about 20 to about 42⁰C, and/or is inactivated when heated to high or extreme temperatures, such as temperatures over 65⁰C; is, or is substantially homologous to, a native protein, such as a native mammalian or human protein and not expressed from nucleic acids in concatameric form, where the promoter is not a native promoter in to the host cell used in the array but is derived from another organism, such as E. coli.

Kits

The present invention also provides kits useful for expression and extracellular secretion of a heterologous protein or polypeptide of interest. The kit comprises one or more of the improved Pseudomonas fluorescens -dcnycd host cell populations described herein. These kits may also comprise reagents sufficient to facilitate growth and maintenance of the cell populations as well as reagents and/or constructs for expression of a heterologous protein or polypeptide of interest. The populations of host cells may be provided in the kit in any manner suitable for storage, transport, and reconstitution of cell populations. The cell populations may be provided live in a tube, on a plate, or on a slant, or may be preserved either freeze-dried or frozen in a tube or vial. The cell populations may contain additional components in the storage media such as glycerol, sucrose, albumin, or other suitable protective or storage agents.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL EXAMPLES

Materials and Methods

Construction of E. coli Alkaline Phosphatase Expression Plasmid pDOW2299

The DNA coding sequence for the E. coli alkaline phosphatase gene, PhoA, was obtained from plasmid pDOW 1322-1 which contains the cloned PhoA sequence (Xbal-Xhol). After a double-digest of the pDOWl 322-1 plasmid with Xbal and Xhol enzymes (New England Biolabs), the 1.4 kb PhoA fragment was purified by agarose gel extraction using the Qiagen Gel Extraction Kit (Qiagen #28704) and then ligated into Spel-Xhol digested pDOWl 169 using T4 DNA ligase (New England Biolabs). The ligation mixture was transformed into electrocompetent P. βuorescens strain DC454 cells and then plated onto glucose agar. Transformant colonies were screened by plasmid prep (Qiagen #27104) and restriction digestion of plasmid DNA isolated from shaking cultures grown for ~16 hours in 3 ml mineral salts medium at 30⁰C. Positive clones that were confirmed by DNA sequencing of the plasmid insert were designated as pDOW2299.

DNA Sequencing Clones were analyzed by sequencing using Big Dye version 3.1 (Applied

Biosystems). Reactions consisted of 2 μl of sequencing premix, 1 μl of 6.4μM primer, 50 fmol of DNA template, 3μl 5X buffer + H₂O to adjust volume to 20 μl. Sequencing reactions were then purified using G-50 (Sigma) and loaded into the ABI3100 sequencer. Sequence data were assembled and analyzed using the Sequencher™ v4.7 software (Gene Codes Corporation).

EMS Mutagenesis ofP.fluorescens Strain DC454/ pDOW2299

Chemical mutagenesis of the P. βuorescens strain DC454 cells harboring the E. coli alkaline phosphatase expression plasmid pDOW2299 was carried out using ethyl methane sulphonate (EMS) (Sigma #M0880-5G). DC454/ pDOW2299 cells were grown in a glucose medium at 30° C for 16 hours in a 250 ml bottom-baffled flask (reaching an ODβoo of 4.8). The culture was spun down (6,000 X G) in a 50 ml screw cap Falcon tube for 15 minutes to harvest the cells. The cell pellet was then resuspended to a 20 ml volume in mineral salts medium and then divided in half, with each 10 ml of culture placed in a separate 50 ml screw cap falcon tube. Another 10 ml of glucose medium was added to each culture (for a final volume of 20 ml). EMS solution was then added to one of the 20 ml cultures to a final concentration of 2% EMS. Both cultures (+ EMS and - EMS) were allowed to grow with gentle shaking at 30⁰C and 2 ml samples were withdrawn at 1, 2, 3, 4 and 5.5 hours incubation. After withdrawal, each sample was spun to pellet the cells in 2 ml microfuge tubes for 1 minute at 14,000 rpm. The pellet was resuspended in 2 ml of glucose medium and spun again. After repeating the wash twice more, the final pellet was resuspended in 850 μl mineral salts medium to which was added 150 μl of 80% glycerol. Finally, 100 μl of the glycerol suspension was aliquotted into separate 0.5 ml microfuge tubes and frozen immediately at -80° C. Viable counts for each time point were determined by dilution plating of the glycerol stocks and comparing the colony counts from the culture treated with EMS versus the untreated culture. From the viable count analysis, it was determined that cells incubated at 3 hours (T=3) with 2% EMS produced a 0.1% survival rate. It was also observed that the T=3, EMS treated culture produced colonies that varied in size and colony morphology, which indicated successful mutagenesis.

High Throughput Growth of Mutants The mutant cells (T=3 glycerol stocks) were plated on a salts media with 1% glucose in 245 mm square Bio-Assay dishes (Corning # 431272) and grown at 30⁰C for 48 hours. To start the seed plate cultures, colonies were picked into 96 well 2 ml deep well plates containing 900 μl of Soy LB medium using the Genetix QPix robot. Plates were covered with gas-permeable adhesive seals (ABgene # AB-0718) and incubated in a Kuhner incubator/shaker at 3O⁰C, 300 rpm (50 mm diameter throw) and 75% humidity. Twenty-four hours after inoculation of the seed plates, the seed plate cultures were used to inoculate the expression plates. Inoculation of the expression plates utilized the Tecan program "Inoculation " to transfer ten microliters of each seed culture into corresponding wells of another 96 well deep well plate containing 500 μl of HTP -YE medium. Two wells in each seed plate were left empty to manually add a negative and positive control to the expression plate at this time. Plates were returned to the incubator for growth as described above. The seed cultures were diluted and samples were then read for optical density at absorbance 600 nm using the Molecular Devices SpectraMax Plus 384 machine with SOFTmax Pro 3.1.1 software. Expression cultures were induced 24 hours after inoculation, adding 15 μl of 10 mM IPTG to each well. Cultures were also sampled for OD₆oo measurements of the IO timepoint as described above and then returned to the incubator. Twenty- four hours post-induction (124), plates were once more sampled for ODβoo measurements and then sampled for the MUP activity assay. Samples were spun down in a Eppendorf tabletop centrifuge at 4,000 rpm for 10 minutes to pellet the cells. Subsequently, 125 μl of cell free broth samples were transferred into a 96 well 0.45 um filter plate. Vacuum was applied to filter the cell free broth samples into a 96 well 1 ml plate; filtrates were covered with foil adhesive seals and frozen at - 20⁰C until assayed.

Expression cultures were induced 24 hours after inoculation using the Tecan robot in which 15 μl of 10 mM IPTG was added to each well. Cultures were also sampled for OD₆oo measurements of the 10 timepoint as described above and then returned to the incubator. Twenty-four hours post-induction (124), plates were once more sampled for ODβoo measurements and for cell-free culture broth which was subsequently utilized for the MUP activity assay. Cell-free culture broth samples were collected after the 96 well culture plates were spun down in a Eppendorf tabletop centrifuge at 4,000 rpm for 10 minutes to pellet the cells. The Tecan robot transferred 125 μl of cell free broth samples into a 96 well 0.45 um filter plate. Vacuum was applied to filter the cell free broth samples into a 96 well 1 ml plate; filtrates were covered with foil adhesive seals and frozen at -20⁰C until assayed.

Alkaline Phosphatase Activity Measurements/ Fluorescence Assay

Filtered cell free broth samples obtained from HTP culture growth were measured for alkaline phosphatase activity using the Spectramax GeminiXS

Fluorimeter plate reader (Molecular Devices) set at 360 nm excitation, 449 nm emission and 420 nm bandpass. Reaction mixtures consisting of 25 μl of cell free broth, 165 μl water, and 10 μl of 1 OmM 4-methylumbelliferyl phosphate (MUP-free acid) were incubated at 30° C for 20 minutes in 96 well plates before the relative fluorescence units (PvFU) of the mixtures were measured in the plate reader. MUP is a substrate for alkaline phosphatase that, when hydrolyzed, produces a fluorescent product. The 20 minute-endpoint fluorescence assay was developed by spiking Bacterial Alkaline Phosphatase (PhoA) into DC454/ pDOWl 169 (empty vector) filtered cell free broth samples in order to determine both the linear range of the fluorimeter and the optimal incubation time using 1OmM MUP substrate in the reaction mix.

Plasmid Curing The removal, or curing, of plasmid DNA from mutant strains was facilitated by using 5-fluoroorotic acid (5-FOA) to select against cells harboring plasmids expressing the pyrF gene selection marker, which is used to complement the host cell's uracil auxotrophy. 5-FOA is toxic to cells that can synthesize the pyrF gene product, pyrimidine biosynthetic enzyme orotidine-5 '-monophosphate decarboxylase, and are therefore unable to grow on media supplemented with 5-FOA. Briefly, a single colony was used to inoculate 50 ml of LB (soy hydro lysate) medium supplemented with uracil (750 μg/ml) in a 250 ml bottom-baffled flask which was then incubated at 30° C for 24 hours with shaking (300 rpm). After incubation, 50 μl of culture was spotted onto LB (soy hydro lysate) agar plates supplemented with 250 μg/ml uracil and 500 μg/ml 5-FOA (Zymo Research #F9001-5). Single colonies were produced by dilution- streaking the spotted culture and incubating the plates at 30° C for -48 hours. Single colonies picked from the 5-FOA selection plates were then patched to both glucose agar plates supplemented with 250 μg/ml uracil and to glucose agar plates without uracil in order to identify isolates that could not grow without uracil supplementation and had therefore lost plasmid containing the pyrF selection marker. Plasmid preparations of 5-FOA tolerant strains were also utilized to confirm the loss of plasmid DNA.

Mutant Evaluation Scheme Mutant strains isolated in the initial screen became candidates for evaluation at the 2OL scale after a series of 96-well HTP medium evaluations as summarized in Figure 1. Cell-free broth samples from P.fluorescens mutant library colonies grown and induced in 96-well plates were assayed for alkaline phosphatase activity (as described above). Mutant isolates exhibiting at least a 5-fold (5X) increase in alkaline phosphatase activity compared to the negative/ wild-type control strains in the same plate were noted and re-streaked to glucose plates in order to obtain single colonies. Four single colonies from 5X or better activity mutants were then re-grown in 96-well HTP medium and assayed again for activity. Mutant isolates exhibiting reproducible growth and activity were then cured of plasmid DNA (as described in Materials and Methods). Cured mutant isolates were then re-transformed with pDOW2299 plasmid {phoA expression) and re-examined in 96-well HTP medium for reproducibility of growth and alkaline phosphatase activity in the cell-free broth (four transformant colonies prepared from at least two cured single colony isolates were examined). Re- testing mutant isolates with naive pDOW2299 plasmid was to ensure that the higher alkaline phosphatase activity observed in the cell-free broth from the parent mutant strains was not a result of mutations occurring in the plasmid DNA. Mutants again exhibiting reproducible growth and activity were then examined at the 2OL scale (as described below) for stability of growth and expression of alkaline phosphatase.

2OL Growth Evaluations of Mutant Strains

The inocula for the fermentor cultures were generated by inoculating a shake flask containing 600 ml of a chemically defined medium, supplemented with trace elements, yeast extract, and glycerol with a frozen culture stock. After 16-24 hr incubation with shaking at 32 ⁰C, the shake flask culture was then aseptically transferred to a 2OL fermentor containing a medium formulated to support the production of greater biomass. Dissolved oxygen was maintained at a positive level in the liquid culture by regulating the amount and rate of sparged air and the mixing provided by the agitator, and the pH was maintained at the desired set-point through the addition of aqueous ammonia. The cell number was approximated by determining the optical density of the culture (λ = 575 nm). The fed-batch high density fermentation process consisted of an initial growth phase of ~24 hr, followed by a gene expression phase in which IPTG (0.1 mM final concentration) was added to initiate target gene expression for another 24 hours, during which appropriate samples were withdrawn from the fermentor for various analyses to determine cell density and the level of target protein expression. For SDS-CGE analysis, 2X 0.1 ml of whole broth samples were centrifuged to separate cell-free broth from the cells. Both whole broth and cell free broth samples were then frozen in preparation for subsequent analysis by SDS-CGE.

Analysis by Capillary Gel Electrophoresis (CGE)

Samples were analyzed in a 96-well micro-plate format using the LabChip 90 instrument with the HT Protein Express 200 chip (Caliper Life Sciences). BSA protein standards from 300 to 37.5 μg/mL were run in parallel with test samples assayed at neat, 2-fold, and 4-fold dilutions in phosphate buffered saline. Samples were prepared according to the LabChip 90 protocol. Briefly, in a 96-well polypropylene conical well PCR plate, 4 μL of test sample or standard was mixed with 14 μL of denaturing sample buffer containing DTT, heated at 95 ⁰C for 5 min and diluted by the addition of 70 μL DI water.

Results and Discussion

P. βuorescens Mutant Library Screening The P. fluorescens strain DC454 harboring the E. coli alkaline phosphatase

(PhoA) expression plasmid was subjected to chemical mutagenesis in order to produce a mutant library of cells that were subsequently screened (Figure 1) for an increase in extracellular PhoA activity compared to the parent strain. Plated mutant library colonies were picked and cultured in 96-well plates in HTP medium. After induction of PhoA production for 24 hours, measurements of both the optical density (OD600) of the cultures as well as PhoA activity of filtered, cell-free broth were collected using high-throughput methods. Individual isolates exhibiting increased extracellular PhoA activity compared to the DC454 parent strain cultured in the same plate were noted. Examples of typical results are presented in Figure 2. Mutant isolates exhibiting at least a 3-fold increase in PhoA activity compared to the parent control and showing no significant loss in optical density were noted. Generally, it was assumed that isolates displaying high extracellular PhoA activity along with a significant loss in optical density were exhibiting cell lysis (thus releasing cellular PhoA) and were therefore disregarded for further analysis. Lysed cultures were routinely confirmed by observation of viscous broth and accumulation of precipitate. Using the high-throughput methods described in Materials and Methods, approximately 32,000 mutant colonies were screened utilizing a maximum of 24 96- well plates (2,250 colonies) per week. Of the 32,000 colonies screened, 212/ 32,000 (0.7%) exhibited at least a 3-fold increase while 64/ 32,000 (0.2%) exhibited at least a 5 -fold increase in extracellular PhoA activity. A total of 64 mutants exhibiting at least a 5-fold increase in PhoA activity compared to the parent were subsequently re- streaked from 96-well glycerol stocks in order to produce single colonies. Four isolated colonies for each strain were then re-grown and re-assayed to test for reproducibility of growth and extracellular PhoA activity. Multiple transformants of more than one cured isolate for each strain were then re-grown and re-assayed in order to examine reproducibility with naϊve PhoA expression plasmid. The pDOW2299 plasmid was removed and re-introduced into the mutant isolates in order to ensure that the increased extracellular PhoA activity observed was not a result of mutations originating from plasmid DNA. The HTP 96-well growth plots and cell- free broth PhoA activity assay results for a representative number of the cured and re- transformed mutants examined are shown in Figure 3. It is notable that while a number of the re-transformed mutants did not exhibit extracellular PhoA activity as high as shown before plasmid curing (e.g. P176-D9, P235-C3, P247-B7, P281-E3, and P336-H11) all exhibited increased activity compared to the native control strains grown in the same plate.

Of the 64 total 5 -fold increase mutants re-evaluated using this scheme, 16 showed reproducible PhoA activity in cell-free broth as well as stable growth at every step. While most of these 16 mutants appear comparable in terms of HTP medium growth, some exhibit differences in growth and morphology on plated medium. On glucose plates, the P91-A4 (DC813) and P341-F5 (DC846) strains in particular, produced very small, dry colonies exhibiting slower than normal growth.

Evaluation of Mutants at 2OL scale Sixteen mutant isolates that exhibited reproducible growth and extracellular

PhoA activity at small-scale screening were examined at the 2OL growth scale in order to evaluate PhoA expression and stability of growth in high cell density fermentation. While a number of the mutant strains grew comparably to the DC752 native control strain (non-mutagenized DC454/ pDOW2299), most of the mutants exhibited a range of patterns in growth rates. Strains DC751, DC813 and DC815, in particular, exhibited very long growth phases. Figure 4 shows the PhoA soluble yields before induction and 24 hours post-induction for a representative set of mutants. Plots of total culture soluble PhoA in g/L were compared to PhoA protein (g/L) detected in whole culture cell-free broth (CFB) samples. Similar to the growth plots, all 16 mutants exhibited a range of total soluble and CFB PhoA accumulation. Based upon growth and CFB PhoA accumulation, the top three mutants identified were DC843, DC825, and DC751.

Bar plots comparing CFB PhoA yields vs. whole culture PhoA yields in g/L for 24 hours post-induction samples are provided in Figure 5. While the CFB PhoA yield plots in Figure 4 were not corrected for sampling volume, the summary of protein yield data shown in Figure 5 provides the corrected CFB yields at 24 hours post-induction assuming 40% solids. When error bars are exhibited, bar graphs indicate average yield for all tank runs and error bars indicate standard deviation. The DC843 mutant produced a 6.9-13.4 fold increase in CFB PhoA accumulation compared to the native strain (DC752) runs, while the DC825 and DC751 mutants produced 7.4-8.8 and 3.8-6.5 fold increase in CFB PhoA, respectively. In one particular run, 071016B, the DC843 mutant produced a CFB PhoA yield of 5.1 g/L, which, when compared to the soluble whole broth yield of 5.53 g/L, represents 92% of the total soluble PhoA produced from the cells. In all three DC843 tank runs, the percentage of total PhoA secreted from the cells ranged from 49% to 92%. The DC825 mutant ranked second in this regard, secreting 41% to 71% of total PhoA protein from the cell with the highest CFB PhoA yield of 3.36 g/L. The DC751 mutant also is notable for producing a 3.8-6.5 fold increase in CFB PhoA accumulation (high yield of 2.46 g/L CFB PhoA) compared to the native strain, however, the DC751 mutant not only exhibited a longer growth phase than the DC843 and DC 825 mutants but also produced a considerably more viscous broth at 24 hours post-induction. In addition, CGE gel-like images of the 24 hours post-induction DC751 samples (data not shown) exhibit an increase in cellular protein contamination, or higher background, compared to the other strains, which, together with the increase in broth viscosity, indicated a higher degree of cell lysis. Alternatively, the DC843 and DC825 mutants produced comparable growth to the native strain with no detectable difference in background from the CGE analysis.

In order to examine extracellular secretion of a recombinant protein other than PhoA, the plasmid cured version of the DC825 parent strain, DC831, was examined at 2OL for expression of the Gal 13 diabody protein fused to the pbp secretion signal peptide (expression plasmid pDOW3802). The growth of the MID4380 strain (DC831/ pDOW3802) shown in Figure 6 showed little difference to the native control pbp-Gall3 diabody expression strain DC694 (DC454/ pDOW3802). Figure 7 shows the plots of Gal 13 diabody yields from before induction to 24 hours post-induction as determined by CGE analysis of culture samples; the yield data plotted for each strain is the calculated average for all tank runs (4 tanks MID4380; 2 tanks DC694). The MID4380 mutant host strain not only exhibited a 1.5 fold increased yield of total soluble Gall3 protein compared to the native but a 2.5 fold increase in total CFB Gal 13 protein yield.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A composition comprising a Pseudomonas βuorescens-deriyed cell population capable of expressing a heterologous protein of interest, wherein the level of extracellular secretion of said heterologous protein is increased relative to the level of extracellular secretion of said heterologous protein by a control cell population, wherein said Pseudomonas fluorescens-dsήyed cell population has at least one genomic modification relative to the control cell population, wherein said genomic modification is attributable to said increase in extracellular secretion.

2. The composition of claim 1, wherein said Pseudomonas fluorescens- derived cell population is selected from the group consisting of the cell populations deposited as Accession Nos. PTA-8981 and PTA-8982.

3. The composition of claim 1 , wherein said Pseudomonas fluorescens- derived cell population comprises an expression cassette comprising a nucleotide sequence encoding said heterologous protein.

4. The composition of claim 3, wherein said expression cassette further comprises a nucleotide sequence encoding a secretion signal polypeptide operably linked to the nucleotide sequence encoding the heterologous protein.

5. The composition of claim 1 , wherein the genomic modification affects one or more genes involved in outer membrane vesicle (OMV) formation, OMV function, or both.

6. The composition of claim 1, wherein the level of extracellular secretion from the Pseudomonas fluorescens-deήved cell population is at least two times, at least three times, at least four times, or at least five times the level of extracellular secretion from the control cell population.

7. A kit comprising the composition of claim 1.

8. A method of expressing a heterologous protein of interest comprising: a) obtaining a Pseudomonas fluorescens-deήved cell population comprising an expression cassette encoding said heterologous protein of interest; and, b) culturing the cell population under conditions sufficient for expression of said heterologous protein of interest; wherein said Pseudomonas fluorescens-deήyed cell population has at least one genomic modification relative a control cell population, wherein said genomic modification results in an increase in extracellular secretion of the heterologous protein of interest.

9. The method of claim 8, wherein said Pseudomonas fluorescens- derived cell population is selected from the group consisting of the cell populations deposited as Accession Nos. PTA-8981 and PTA-8982.