WO2004039984A1 - Recombinant protein expression - Google Patents

Abstract

The present invention relates to a method for the production of soluble biologically active proteins, comprising the steps of: (i) introducing into a host cell, an expression vector comprising a polynucleotide that encodes a protein of interest and a suitable promoter; (ii) culturing the host cell under conditions that permit exponential growth of the host cell; (iii) altering the culture conditions to achieve a linear reduction in the growth rate of the host cell in the absence of an agent that induces the activity of the promoter; and (iv) purifying the expressed protein.

Description

Recombinant Protein Expression

Field of the Invention

The present invention relates generally to methods of recombinant protein expression, in particular to a simple and inexpensive method for expression of recombinant proteins, which gives augmented yields of soluble, biologically active recombinant protein in a bacterial cell line. More specifically, the invention demonstrates how the methods may be applied to both automated high-throughput screening of protein:protein interactions and shake-flask or fermenter-scale production of recombinant proteins. Background to the Invention

The production of large quantities of biologically-active protein is a crucial requirement in science research and in the manufacture of many therapeutics, e.g. antibody production. Protein production is usually carried out in recombinant expression systems designed to maximise the amount and activity of the target protein.

The choice of recombinant expression system is limited to bacterial (normally E coli), insect, yeast or mammalian. Each system comes with its own set of advantages and disadvantages, mainly related to solubility, activity, yield, post-translational modifications i.e. glycosylation, and more importantly, expense and end usage. The majority of researchers in the field of proteomics choose £ coli as an initial starting point for expression of recombinant proteins.

Bacterial expression is inexpensive, rapid and relatively simple. However, it tends to be marred by the ability of the cells to quickly package expressed proteins into insoluble bacterial inclusion bodies. Although this may seem an advantage with regard to purification, the recovery of active soluble protein from inclusion bodies requires a prolonged procedure of denaturation and dialysis to reform broken disulphide bonds; even then the percentage recovery of active protein can be very low - often as little as 1 %.

Protein expression in £ coli can be performed in a number of different ways using a variety of expression vectors - cytoplasmic, periplasmic, surface display or secretion. Expression may be constitutive or under the control of an environmentally or chemically inducible promoter. Each vector in turn is designed to maximize the yields of soluble recombinant protein produced, with different vectors incorporating known highly soluble proteins as fusion tags, for example maltose-binding protein (MBP), Glutathione S transferase (TSH) and Thioredoxin (Davis etal., Biotech & BioEng., 1999; 65(4): 382-388). However, such tags are normally quite large in size and can prove difficult, expensive and time consuming to remove. Alternative smaller tags, such as HIS, Myc and Flag, may not necessarily promote solubility, but may interfere less with biological activity. In terms of ease of use, detection and subsequent cost-effective purification by immobilised metal affinity chromatography (IMAC), the HIS tag appearstobethefusiontagof choice (Porathef a/., Nature, 1975; 258:598-599). The method of choice to obtain soluble protein, in most cases, is periplasm- directed expression, since the periplasmic environment of £ coli is more conducive to the formation of disulphide bonds. Traditional thinking suggests that the bacterial cytoplasm is not a suitable environment for the expression of recombinant proteins in soluble form (Cornelis, Curr. Opin. Biotechnol., 2000; 11 :450-454). However it has been demonstrated that disulphide bond formation can take place in the cytoplasm of £ coli strains lacking redox enzymes (Prinz et al., J.Biol.Chem. 1997; 272(25): 15661 -15667), e.g. thioredoxin reductase; such bacterial strains include BL21 DE3 trxB and Origami DE3. The yields of soluble recombinant protein in many bacterial models have so far been disappointing, but in many cases direct extraction of soluble, biologically active protein is considered worth the sacrifice, as refolding is tedious, costly and often unreliable. Moreover, biological activity perse in these systems is an issue that many investigators have failed to address, or wished to avoid.

US Patent No. 5726039 describes an expression system for the production of soluble proteins. The system relies upon an expression vector that utilises a promoter that is activated at low temperature. A host cell, incorporating the expression vector, is cultured under normal conditions (37° C) to promote cell growth and then the cells are transferred to a culture medium at a reduced temperature (15°C - 20°C) to activate the promoter. Although this system results in active soluble protein the soluble expression levels tends to be low due to the chronic activity of the cold shock promoter which results in an accumulation of insoluble aggregates.

Vasina et al., Protein Expression and Purification, 1997; 9:211-218 also describes an expression system utilising so-called cold-shock promoters. There is also a disclosure of using the tac promoter in the presence of the inducing agent IPTG. The requirement for IPTG is a disadvantage for large-scale protein production, where large concentrations of this expensive reagent is required.

There is therefore a need for an alternative expression system that avoids the need for expensive reagents and which can produce active soluble protein under conditions that permit large-scale manufacture. Summary of the Invention

The present invention is based on the realisation that limiting the rate at which recombinant protein accumulates in the cytoplasm of a host cell can improve the yield, solubility and biological activity of the product significantly and that this can be achieved by the linear reduction in the metabolism of the host cell.

According to a first aspect of the invention, a method for the production of soluble biologically-active protein, comprises the steps of:

(i) introducing into a host cell, an expression vector comprising a suitable promoter and a polynucleotide that encodes a protein of interest;

(ii) culturing the host cell under conditions that permit exponential growth of the host cell;

(iii) altering the culture conditions to achieve a linear reduction in the growth rate of the host cell in the absence of an agent that induces the activity of the promoter; and

(iv) purifying the expressed protein.

The method of the invention permits the production of recombinant proteins with augmented yields (mg/L), which have biological activity comparable to native proteins. The methods are suitable for a number of systems, such as automated high-throughput screening of protein-protein interactions and the identification of novel binding candidate ligands, and also large-scale (shake- flask or fermenter) expression of purified recombinant proteins for research or industrial applications.

Using the method, the yields of soluble recombinant protein are significantly augmented. Moreover, the protein is biologically active. Description of the Drawings The present invention is described with reference to the accompanying figures wherein:

Figure 1 is a graphic representation of a chromatogram showing the amount of purified protein isolated after culturing a recombinant cell according to the present invention; Figure 2 is a graphic representation of a SDS PAGE gel, where lane 1 represents protein standards, and lanes 2, 3 and 4 represent 5, 10 and 25 μl samples of IMAC purified cell lysate, respectively;

Figure 3 is a graphic representation of the amount of protein produced according to the invention; Figure 4 shows the results of an ELISA experiment carried out in a 96 well plate;

Figure 5 represents the results of a dot dot experiment, where (A) represents filtrates of total protein from cells lysed under non-denaturing conditions prior to filtration and (B) represents filtrates containing the soluble protein from cells lysed under non-denaturing conditions;

Figure 6 represents the results of an ELISA experiment;

Figure 7 is a graphic representation of the levels of binding affinity of anti- CRP G8 lysate for its target antigen;

Figure 8 is a graphic representation of the purification of recombinant anti- CRP binding protein;

Figure 9 represents an SDS PAGE gel stain, where lane 1 represents the protein markers, lane 2 represents crude lyrate, and lanes 3 to 7 represent IMAC sample (2.5 μl, 5 μl, 7.5 μl, 10 μl and 15 μl, respectively); and

Figure 10 is a graphic representation of ELISA results for the recombinant anti-CRP clone G8.

Description of the Invention

The present invention allows the selection of a carefully controlled set of conditions that permits efficient expression of soluble, biologically-active recombinant protein in the cytoplasm of a host cell.

The host cell may be any cell suitable for expression of a recombinant polynucleotide. Bacterial cells are preferred, in particular £ coli cells. The method of the invention provides a simple, rapid method for the production of active soluble recombinant proteins. Typical yields from a litre of shake-flask culture are 5-10 mg. The potential yields for fermenter scale cultures can be up to 10-fold greater in some cases, as conditions can be controlled more carefully. The method of the invention relies on the alteration of the growth conditions of a host cell to reduce the growth rate at which protein accumulation occurs in the cytoplasm. There are many ways in which the growth rate may be reduced including a reduction in the availability of a carbon/energy source, or decreasing the availability of oxygen to the host cells. The preferred way to reduce the growth rate is to lower the temperature of the culture.

A requirement of the present invention is that the reduction in metabolic growth is achieved gradually. A linear reduction of the growth rate avoids "shocking" the cells and achieves high levels of protein production. A linear reduction in the growth rate can be achieved by appropriate alteration of the culture conditions. If temperature is needed to control the growth rate, the reduction in growth can be achieved by the gradual incremental reduction of the temperature over time. However, it is possible to achieve the linear reduction simply by reducing the temperature of the fermenter to a required low temperature (e.g. 16°C - 20°C) and allowing the culture to decrease in temperature naturally over time. Incremental reductions in the temperature of the fermented are therefore unnecessary, as the culture medium will gradually decrease in temperature to arrive at the new low temperature. This is different from transferring the cells into a now culture medium which is at a pre-set low temperature designed to "shock" the cells. The reduction in the growth rate is said to be "linear". This is to be interpreted as meaning that the growth rate decreases at a substantially constant rate over time. This may be carried out until a desired lower growth rate is achieved, when the growth rate may be stabilised.

In one embodiment, the temperature of the cell culture is maintained from 30°C to 40°C to achieve growth of cells, and the temperature is then reduced to below 25°C, more preferably reduced to below 10°C. This reduction in temperature may be carried out over a time course of at least 1 hour, more preferably over a period of at least 10 hours, and most preferably over a period of at least 15 hours.

The growth rate may be monitored using conventional methods known to those skilled in the art, including spectroscopic analysis. The reduction in the growth rate may be carried out until a suitable level of soluble protein expression is achieved. The culture conditions may then be maintained at a constant level while protein production occurs.

Expression in the bacterial cytoplasm is often under the control of an inducible promoter. Examples of these are lac, tac or trc which vary in their ability to regulate the expression of said proteins after induction, normally by IPTG (1 ). However, comparative assays have been carried out by the inventors, where IPTG (chemical induction agent), was added or omitted. It was demonstrated that activation of the T7 promoter with IPTG at low temperature (16°C) had no effect on the total yield of soluble recombinant protein, however, the protein was biologically inactive. This suggests that the addition of IPTG to the culture at lower temperature induces a significant shift in the protein expression kinetics. Thus, the addition of IPTG may increase the V_max of protein expression, allowing rapid synthesis of the recombinant protein. However, this change in kinetics may result in the production of biologically inactive protein. Accordingly, a combination of reduced temperature and the absence of an induction agent, favours correctly folded biologically-active soluble recombinant protein.

The methods presented herein show that expression of soluble recombinant protein with higher biological activity can be achieved in cultures maintained at low temperature in the absence of inducible agent (IPTG). Such a system has direct benefits in terms of yield, minimal operator input and environmental safety and a direct saving in terms of cost. Without wishing to be bound by theory, the soluble expression of protein may be related to the metabolic rate of the cells during the period of soluble expression using optical density.

The following relationships may apply: E_s (soluble expression) is inversely proportional to incubation temperature

(T)

E, (insoluble expression) is proportional to incubation temperature (T)

Therefore:

E_s = k (1/T) (where k is a constant) Ej = k,T (where k, is a constant)

When this is related to the optical density at 600 nm (OD₆₀₀), the following equation can be constructed, where ΔOD₆₀₀ is the change in optical density at 600 nm between times t=0 and t=16 when OD₆₀₀ < 0.2-0.3 hours after decreasing the temperature to 16°C. t= 16

E_s = Ei.k.^/T. [ΔOD₆₀₀] t= o It is preferred to achieve an OD₆₀₀ of less than 0.5, more preferably less than 0.3, and then decrease the growth rate for the production of soluble protein. The method of the invention can be carried out using any suitable expression vector. It is preferable that a cytoplasmic or periplasmic expression vector is used. Suitable expression vectors are known in the art, and include: but are not limited to pRSET (Invitrogen), pET vectors (Novagen), pQE series (Qiagen).

The expression vector comprises a polynucleotide that encodes the protein of interest, and also a suitable promoter for initiating expression by directing the binding of RNA polymerase. Suitable promoters will be known to the skilled person and include: T7 lac, T7 tac etc.

The expression vector may comprise additional components, including control elements, initiation codons (if not present on the protein-encoding polynucleotide), and a ribosome binding site, as will be appreciated by the skilled person. The expression product can be any protein of interest. Typically, the protein will be a therapeutically useful protein, including enzymes, antibodies, hormones etc. The protein will usually comprise more than 10 amino acids.

The reference to "protein" or "protein of interest" is intended to encompass peptides and chime c proteins/peptides. The protein will usually derive or originate from an eukaryotic source.

It will be evident to the skilled person how to construct a suitable polynucleotide that encodes a protein, into the expression vector.

A general summary of the method of the invention is as follows. Cells transformed with a cytoplasmic expression vector containing, for instance, a T7 promoter, typically pRSET (Invitrogen), or others known to those skilled in the art, can be seeded at low density into an appropriate volume of culture medium and incubated at 37°C until the OD_600nm reaches between 0.2-0.3 (1 cm path length). The incubator temperature is then reduced to 16°C and the culture allowed to equilibrate to this temperature. At this point it is conventional to induce expression by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG), but in this invention the agent should be omitted from the procedure. Incubation at 16°C is continued for up to a further 16 hours, at which time the cells are harvested either by centrifugation (for large volumes) or vacuum filtration (microtiter plate cultures). A proprietary non-denaturing lysis buffer containing nucleases can then be added, and the cell suspension incubated with mixing at 4°C for 1 hour. The lysate can then be clarified either by centrifugation (for large volumes) or vacuum filtration (microtiter plate cultures).

Large-scale cultures usually progress to purification by immobilized metal affinity chromatography. No single metal is universally applicable for the purification of all recombinant proteins, so at this point small-scale tests can be carried out to determine the optimal metal for purification of each recombinant protein product. Thereafter the recombinant proteins can be purified using the metal chelate and by gel permeation chromatography (see Example 1 below). The method can be used with any bacterial cell as the host. However, £ coli strains lacking redox enzymes are preferred, e.g. BL21(DE3) trxB and Origami DE3. The invention will now be further described in the following non-limiting Examples. Example 1

This example describes the amplification of a gene of interest, e.g. human fatty acid binding protein (FABP), amplified from a source of human cardiac tissue cDNA. The amplified gene was cloned into the pRSET vector (Invitrogen, UK) containing a N terminal HIS tag, by conventional cloning methods and expressed and purified using the methods referred to in this manuscript. Transformation BL21 DE3 trxB cells (Novagen) were freeze-thawed on ice (10-15 minutes). Prepared vector containing the DNA sequence (2μl/vial) was then added to the cell suspension and the mixture incubated on ice for 1 hour. The cells were then heat shocked at 42°C (2 minutes) and then incubated on ice for 2 minutes. SOC medium (Novogen) (250μl) was then added to the cells at room temperature and the mixture incubated at 37°C with mixing for 1 hour. The cells were then plated on agar (containing 15 μg/ml kanamycin and 50 μg/ml ampicillin) and grown overnight at 37°C. Growth Conditions

Bacterial colonies were removed from the culture plate and used to inoculate 10 ml LB broth (Sigma). Incubation was continued overnight at 37°C. This culture was then used to inoculate 1L LB broth, pre-equilibrated to 37°C (this is an important step to avoid growth arrest). The culture was then incubated at 37 °C with shaking until an OD_600nrn of 0.2 to 0.3 was reached. At this point, the incubator temperature was reduced to 16°C and the cells held in the incubator over this change in temperature. The cells were incubated at this temperature (16°C) with shaking in the absence of any induction agent for up to 16 hours. Protein Expression

Cells were pelleted by centrifugation (5000rpm, 30 minutes, 4°C) and the culture media discarded. The resulting cell pellet was then snap frozen in liquid nitrogen, stored on ice and then solubilised in lysis buffer (10 ml lysis buffer/1 L culture) (sodium chloride 300 mM, sodium dihydrogen phosphate 50 mM, glycerol 10%, BugBuster 10X(1 ml/10 ml buffer), benzonase (10000U) (1μl/ml) pH 8.0) by gentle mixing to avoid unnecessary frothing. Cocktail inhibitors (Sigma) were added (1 ml/30 g wet weight cell pellets) and the suspension mixed for 1 hour at 4°C. Cell lysate was clarified by centrifugation (10 OOOrpm, 30 minutes, 4°C). Purification Methods Immobilised Metal Affinity Purification (IMAC)

The cell lysate was first filtered through a 0.45μ filter to remove cell debris. A metal chelate test was then conducted to evaluate binding efficiency for the target recombinant HlS-tagged protein (1 ml/injection) (the efficiency of purification of a given protein by IMAC is highly dependent on the chosen metal chelate, therefore each expressed protein requires an independent metal test before large scale purification). The cell lysate was injected unto a BioCAD 700E chromatography system. Captured HIS tagged protein was then eluted using imidazole (150 mM) or an imidazple gradient (1 to 250 mM). Gel Permeation Chromatography

Eluted IMAC samples were initially concentrated to 500 μl using a Vivaspin column prior to injection onto a HR75 Superdex column. Proteins were separated using a flow rate of 0.66 ml/min and eluted in HEPES buffer. Results

1. Purification and Quantification of Expressed Protein.

Figure 1 shows the IMAC purification profile for fatty acid binding protein (FABP). The Bradford Assay

was then used to evaluate the total recombinant protein recovered following GPC purification using the methods described.

2. SDS PAGE

Figure 2 demonstrates the expression of recombinant fatty acid binding protein FABP following IMAC purification (nickel chelate). Samples were analysed on a 12.5% SDS PAGE gel, which was then stained with Coomassie Blue and destained overnight. 2. ELISA

ELISA evaluated recombinant protein expression (FABP) (figure 3). FABP (100, 50, 25, 12.5, 6.25, 3.125, 1.6 and 0.8 ng/well) was prepared in PBS (pH 7.0). FABP samples (100μl/well) were added to each well in triplicate and incubated for 1 hour at 37°C. The wells were then washed (x1) on a plate washer with TBS containing 0.1 % Tween 20 (TBS-T). Wells were then blocked for 1 hour with a blocking solution (3% casein in maelic acid) (300 μl/well) for 1 hour at 37°C. The wells were again washed (x1) with TBS-T. Commercially available anti-FABP HRP (1:1000) was prepared in 3% Roche block in TBS-T. This was added to wells of the ELISA plate (100 μl/well). The plate was then incubated for a further 1 hour at 37 °C. The plate was again washed (x6) and 10Oμl of TMB added. The plate was covered and incubated at RT for 10 minutes and the reaction terminated by the addition of 2N sulphuric acid (100 μl/well). The ELISA plate was then analysed at an absorbance of 450 nm. The results demonstrated a linear increase in signal with respect to antigen (Figure 3). Example 2

This Example describes a fully automated pilot scale robotic assay capable of producing soluble, biologically active recombinant ScFv proteins isolated after a particular selection technique, from the cytoplasm of £ coli. The assay is directly scalable in terms of the number of colonies to screen per day, which is limited only by the scale of automation available. The volume of culture is also directly scalable for the production and purification of any amount of active protein for the required application. Amplification and Cloning ofScFv Sequences Preselected or naive ScFv DNA fragments were amplified by PCR using

Taq polymerase and sets of gene specific primers. Primers were based on those designed by Sblattero & Bradbury Immunotechnology, 1998; 3(4):271-278 and modified to introduce a Nco I site at the 5' end and a Not-I site at the 3' end of each sequence. PCR products were restricted and ligated into the £ coli expression vector pRSET, containing a N terminal HIS tag (Invitrogen) using the restriction sites introduced with the primers. Ligated plasmids were freshly transformed into the competent £ coli strain BL21 DE3 trxB (Invitrogen), plated onto LB agar containing 50 μg/ml ampicillin and 15 μg/ml kanamycin and incubated overnight at 37 °C. Growth in 96 well Microtitre Plates and Harvest into 96-Well Filter Plates

Individual colonies were picked and arrayed into microtitre wells containing 300 μls of LB broth media supplemented with 50 μg/ml ampicillin and 15 μg/ml kanamycin using a colony picker. Plates were transferred to the robotic deck using a gripper arm and placed onto the purpose built shaking 37 °C platform. During incubation, OD readings (600nm) were taken every 20 minutes using an integrated plate reader (Spectracount, Packard Biosystems) and plate averages calculated automatically. Once OD readings across the plate reached between 0.250 and 0.300 (adjusted to 1 cm light path), 50 μl of each culture were subsequently removed and used to seed fresh microtitre wells containing 250 μl of LB broth media supplemented with 50 μg/ml ampicillin and 15 μg/ml kanamycin. Duplicate plates were removed from the robotic deck and incubated overnight in a stand alone shaking 37 °C incubator, supplemented with 15% v/v glycerol and stored at -80°C. After an initial 1 hour cooling, the remaining microwell culture plates were transferred to a dedicated shaking platform maintained at 16°C and incubated for up to 16 hours.

Aliquots of each culture (200 μl) from each well of the microwell plate were transferred to 96 well multiscreen DV durapore filter plates with 0.65 μm pore size (Millipore) using a dedicated liquid handling system. Culture medium was removed by vacuum filtration using an integrated vacuum manifold and the retained cell pellet subjected to lysis directly in the filter plate. Lysis of Culture Pellets on 96-Well Filter Plate Proprietary non-denaturing bacterial cell lysis solution (100 μl) (Novagen) was added to each of the wells. Plates were transferred back to the dedicated 16°C platform for 30 minutes with shaking. Filter plates were transferred to the integrated vacuum manifold and soluble ScFv protein separated from insoluble by automated vacuum filtration.

Identification of Soluble ScFv Clones by Direct ELISA

Soluble ScFv lysates were collected into a Labsystems EB ELISA microplate containing 100 μl of carbonate buffer (pH 9.6) per well. Plates were covered and incubated on the shaking 37°C platform for 1 hour. After washing once in TBS-T, using an integrated plate washer, wells were blocked in 1 % BSA in tris buffered saline pH 7.4 (TBS) for 1 hour at 37 °C. Blocking solution was removed and wells probed with a 1 :5000 dilution of anti-HIS HRP monoclonal antibody (Sigma) for 1 hour at 37 °C. Wells were washed six times in TBS-T, again using the integrated plate washer, over a 5 minute period and probed with TMB solution in the dark for 20-30 minutes. 2N sulphuric acid solution was added to stop the reaction and the OD readings read at 450 nm. Dot Blot Analysis of Soluble Clones to Check Expression Levels At random, 96 well collection microwell plates were removed from the robotic deck and lysates assayed for expression levels. Briefly, 0.5μl aliquots of each lysate were removed pre and post filtration and spotted in an ordered fashion onto nitrocellulose membrane. Membranes were air dried at 37°C for 1 hour and blocked with 1% BSA in TBS. After blocking, membranes were exposed to a 1/5000 dilution of anti-HIS HRP monoclonal antibody in 1% BSATBS for 1 hour at room temperature. Membranes were washed 3 x 5 minutes in TBS-T and 1 x 5 minutes in TBS and developed using an enhanced chemiluminescent (ECL) detection kit (Roche). Identification of Soluble Binding Clones by ELISA (Immobilized Antigen) Soluble ScFv lysates were collected in a Labsystems EB ELISA microplate previously coated with human recombinant C-reactive protein (200 ng/well) for 1 hour at 37°C, washed 1X with TBS-T and then blocked in 5% BSA containing 0.2% SDS for 1 hour at 37°C. Wells were washed 6X in TBS-T over a 5 minute period and probed with a dilution of anti-HIS labelled HRP antibody (1/5000) for 1 hourat37°C. Wells were washed 6Xwith TBS-T over a 5 minute period and probed with TMB solution in the dark for 20-30 minutes. 2N sulphuric acid solution was added to stop the reaction and the OD read at 450 nm. Clones displaying an OD above general background were taken to be possible binders. Pilot Scale Expression of Potential Binding Clones from Assay B

Positive clones were identified from the ELISA results and hit picked from the backup plates using the robotic workstation. 50 μl of each culture was used to seed 3 ml of LB broth containing 50 μg/ml ampicillin and 15 μg/ml kanamycin. Cultures were incubated at 37°C until the OD_600nm readings reached 0.2 - 0.250. Cultures were chilled immediately to 16°C for 1 hour and incubated for a total of 16 hours at 16°C. Cell culture was removed and centrifuged at 13000 rpm for 10mins. Spent culture medium was removed and the resulting cell pellet resuspended in 1ml of 1 X proprietary cell lysis solution (BugBuster, Novagen) containing benzonase nuclease (Novagen). Lysis was allowed to continue for 1 hour at 16°C. Lysates were clarified by centrifugation at 13000 rpm for 10 minutes to remove insoluble debris including inclusion bodies and the supernatants removed and stored on ice. Confirmatory ELISA Analysis of Potential Binding Proteins

Labsystems EB microwell ELISA plates were coated with a concentration gradient of the corresponding antigen in PBS (pH 7.4). ELISA plates were sealed and incubated at 37°C for 1 hour. Plates were washed once in TBS-T to remove unbound antigen and blocked with 5% BSA containing 0.2% SDS in TBS for 1 hour at 37°C. Blocking solution was removed and wells probed with neat bacterial lysate for 1 hour at 37 °C. After washing six times over a 5 minute period in TBS-T, plates were probed with a 1/5000 dilution of an anti-HIS HRP antibody (in blocking solution) for 1 hour at 37°C. Plates were washed six times over a 5 minute period in TBS-T and probed with TMB detection solution in the dark for 20-30 minutes. The reaction was stopped by the addition of 2N sulphuric acid and OD read at 450 nm. Results Solubility Screening of ScFv Fragments

Bacterial colonies were picked at random, used to seed culture plates and expressed as previously explained. Figure 4 shows a typical soluble expression pattern for 96 individual clones after disclosure using the anti-HIS HRP monoclonal antibody. The results show that 60-70% of clones containing ScFv sequences can produce protein in a soluble form under these assay conditions. The absence of soluble protein may in part be due to sequence abnormalities as a result of PCR amplifications. The results also demonstrate the. ease of immobilization of ScFv fragments to an inert surface. Dot Blot Analysis

Typical results of dot blot analysis are shown in Figure 5. Approximately 60-70% of the clones are soluble under these conditions. A comparison between total protein and soluble only fractions, demonstrates that the greater proportion of HIS tagged protein produced in each individual sample resides in the soluble fraction.

Identification of Soluble Binding Clones by ELISA (Immobilized Antigen)

Figure 6 shows the typical binding pattern for 96 individual bacterial colonies containing ScFv fragments previously selected against their target antigen during several rounds of ribosome display. Typical ELISA results are shown, with the OD_450nm readings shaded referring to binding clones. The assay demonstrates that 14% of the clones are potential binders to human recombinant C-reactive protein. Pilot-Scale Expression Analysis.

The potential binding clones identified above were hit picked from their corresponding backup plates and used to seed 3 ml expression cultures as previously mentioned. Diluted lysates were applied to wells, coated in various concentrations of target antigen. The graphs in figure 7, show the binding of a selected ScFv to target antigen in a dose dependent manner. These results demonstrate the scalability of this particular assay in terms of the production of soluble, active recombinant ScFv proteins.

Large Scale Expression, Purification and Analysis of a Confirmed Candidate Binding Clone.

Clone G8 (figure 6) was expressed and purified as described in example 1. Figures 8-10 describe the IMAC purification profile, SDS PAGE analysis of recovered soluble protein and a direct ELISA demonstrating binding activity of the GPC purified ScFv fragment to immobilised hrCRP.

Claims

1. A method for the production of soluble biologically active proteins, comprising the steps of:

(i) introducing into a host cell, an expression vector comprising a polynucleotide that encodes a protein of interest and a suitable promoter;

(ii) culturing the host cell under conditions that permit exponential growth of the host cell;

(iii) altering the culture conditions to achieve a linear reduction in the growth rate of the host cell in the absence of an agent that induces the activity of the promoter; and

(iv) purifying the expressed protein.

2. A method according to claim 1 , wherein step (iii) is carried out by the linear reduction of the culture temperature.

3. A method according to claim 1 or claim 2, wherein step (ii) is carried out at a temperature of from 30°C to 40°C, and step (iii) is carried out by reducing the temperature stepwise to below 25 °C.

4. A method according to any preceding claim, wherein step (iii) is carried out by reducing the temperature stepwise to a temperature of approximately 10°C.

5. A method according to any preceding claim, wherein step (iii) is carried out for at least 10 hours.

6. A method according to any preceding claim, wherein the cells are cultured in step (ii) to a maximum OD₆₀₀ ≤ 0.2-0.3.

7. A method according to any preceding claim, wherein the expression vector is a cytoplasmic expression vector.