MXPA96004047A - Increased secretion of polipepti - Google Patents

Abstract

The present invention relates to a peptide signaling sequence and the nucleic acids encoding this sequence, which are useful for improving the efficiency of the secretion of NGF polypeptides. A method for preparing the NGFsin M polypeptides is also provided

Description

INCREASED SECRETION OF POLIPEPTIDES Field of the Invention This invention relates to a novel signaling peptide and nucleic acid sequences useful for increasing the secretion of certain polypeptides. The invention further relates to a method for increasing the efficiency of the secretion of these polypeptides from bacterial cells.

BACKGROUND OF THE INVENTION Direct expression of polypeptides Many polypeptides of pharmaceutical importance are prepared in prokaryotic cells such as bacteria, using recombinant DNA techniques. The DNA that codes for the polypeptide (often obtained from human tissues) can be inserted into the cell "host" wherein it is expressed, (ie translated) from the DNA for the polypeptide in the cytoplasm. The polypeptide is then purified from REF: 23146 host cells. 'This procedure is often referred to as "direct" expression of the polypeptide. Although direct expression of a polypeptide is often the most convenient way of manufacturing the polypeptide, problems associated with this method may exist. For example, as the polypeptide is synthesized in the host cell and begins to accumulate, the polypeptide can become toxic to the host cell and kill it. In addition, intracellular proteases can rapidly degrade polypeptide molecules as they are synthesized. In addition, as the intracellular concentration of the polypeptide is increased, the machinery of the host cell can cease manufacturing via a "feedback mechanism" in the host cell that directs the termination of the synthesis of the polypeptide. Another possibility is that polypeptides that can remain in the cytoplasm after synthesis can be subjected to posttranslational modification such as acetylation.

Secretion of Polypeptides To avoid the problems associated with the direct expression of the polypeptides, other methods have been developed for the expression of the polypeptide. One such method called "indirect expression" allows the polypeptide to be secreted from the host cell as it is produced. This method is also known as "secretion" or "processing" of the polypeptide. After secretion, the polypeptide can be isolated from the culture medium or, in the case of gram negative bacterial host cells, from the periplasmic space or "periplasm", the area between the inner and outer cell membranes. The use of the secretion can therefore help to diminish the problems associated with the intracellular accumulation of the polypeptide. Some polypeptides produced naturally by bacterial cells (and other cells) are secreted from these cells into the extracellular environment (or, in the case of gram-negative bacteria, into the periplasm). The secretion of a polypeptide from a bacterial cell seems to involve the orchestration of several intracellular proteins known as caperone proteins (Zhu et al., Pharm. Tech., April 1993, pp. 28-38, Simonen et al., Microbiol. ., 57: 109-137

[1993]) that identify the polypeptide to be secreted and aid it in the secretory process. The secreted polypeptides typically have the following structure: they contain as a part of the amino acid sequence a signaling polypeptide (also known as leader peptide, leader sequence, or signaling sequence). A signaling peptide is typically a relatively small peptide, and is usually synthesized as the amino terminal portion of polypeptide during translation. The polypeptide with its bound signaling peptide is often referred to as the "precursor polypeptide" or "the immature form of the polypeptide". The signaling peptide directs the full-length peptide through the cell membrane. In the course of secretion of the polypeptide, the signaling peptide breaks down. As a result, only the "mature" form of the polypeptide is secreted through the cell membrane (or the inner cell membrane for gram negative bacteria, as described below). Since the signaling peptide breaks down, the mature polypeptide typically has a smaller molecular weight compared to that of the precursor polypeptides. The nucleic acid sequences, or genes, that code for most of the naturally occurring polypeptides that are intended for secretion typically comprise the DNA sequence for the precursor polypeptide, i.e., the 5 'end of the gene that contains the sequence encoding the signaling peptide, and the 3 'end of DNA of the signaling sequence is linked to the 5' end of the sequence encoding the mature form of the polypeptide. Therefore, during translation, the precursor polypeptide is synthesized as a single unit with the signal sequence in the terminal ring of the polypeptide. Many prokaryotic signaling peptides have now been identified (See for example, Gennity et al., J. Bioenerg, Biochem., 22: 233-26

[1990]). Signaling peptides often share certain structural characteristics. For example, many signaling peptides of prokaryotic origin are approximately 20-30 amino acids in length. In addition, the amino terminus of the signaling peptide is typically positively charged and the central portion of the signaling peptide is typically hydrophobic (Pugsley (Microbiol. Rev., 57: 50-108

[1993]) .Despite these similarities, each of the secreted polypeptides currently identified in some prokaryotic organisms, such as E. coli bacteria, possess a unique signaling peptide sequence.In addition, not all cell types are "recognized" and therefore have the appearance of processing All signaling peptide sequences A particular signaling peptide can be recognized and thus be able to direct a polypeptide through the cell membrane in certain prokaryotic cells, but not be functional in eukaryotic cells. is not secreted by a prokaryotic cell to be secreted using recombinant DNA technology This can be done by creating a nucleic acid construct wherein the DNA encoding the polypeptide is linked at its 5 'end to a natural or synthetic DNA sequence encoding a signaling peptide. For secretion, the sequence of the signaling peptide selected must be one that is recognized by, and by ID both is capable of being processed by, the host cell in which this construct is to be inserted and expressed. Thus, for example, a signaling peptide obtained from a naturally secreted bacterial polypeptide can be linked to a polypeptide from a source such as a human tissue thereby creating a hybrid precursor polypeptide that can be synthesized in, and secreted from, those bacterial (or other prokaryotic) cellular species that recognize and are capable of processing the signaling peptide. The hybrid construct can be introduced into the host cell, and the host cell can then have the ability to manufacture and secrete the polypeptide. A number of factors determine the actual amount of polypeptide that is produced by a bacterial cell - (apart from whether or not the polypeptide is secreted from the cell). Such factors as culture conditions (Jacques et al., J. Mol. Biol., 226: 597-608

[1992]), the speed of translation initiation (Gold, Ann. Rev. Biochem., 57: 199 -233

[1988]), and the rate of degradation of the polypeptide in the cell (due at least in part to the intracellular proteases) also appear to affect the rate and amount of polypeptide that is secreted. It has also been found that the type of residual amino acids present in the amino terminus of the mature polypeptide seems to have an effect on secretion; the secretion seems to be reduced where some of these residues are positively charged (Andersson et al., Proc. Nati, Acad. Sci. USA, 88: 9751-9754

[1991]).

Polypeptides "without Met" Many therapeutically useful human polypeptides are manufactured in prokaryotic cells such as bacteria using recombinant DNA methods. A heterologous polypeptide (such as a human polypeptide) made in a prokaryotic cell using recombinant DNA technology is not always identical to the form of the polypeptide found in nature. The two forms may differ slightly in their chemical structure. For example, many human polypeptides manufactured in bacterial host cells using recombinant DNA technology have the amino acid methionine, "Met", at the amino terminus ("amino terminus Met"). Those same human polypeptides in their naturally occurring form often do not contain the amino terminal Met, since this is usually removed during the secretion of polypeptides from human cells where they are naturally synthesized in the blood stream or other extracellular fluids where they are found naturally. In some cases where human polypeptides are synthesized in prokaryotic cells, the amino terminal methionine can be removed by an aminopeptidase from the cytoplasmic methionine of the host cell, however this removal, or disruption, rarely occurs where the polypeptide is overexpressed in the host cell.

NGF polypeptides The NGF (nerve growth factor) family of polypeptides, or "NGF polypeptides", are a group of structurally and functionally related neurotrophic factors that are found in many cells and tissues of the nervous system and in tissues that are enervated with system components. nervous. Currently, the family of NGF polypeptides comprises the polypeptides BDNF (neurotrophic factor derived from the brain), NGF (nerve growth factor), NT-3 (neurotrophin 3), NT-4 (neurotrophin 4); described in PCT 93/25684 published on December 23, 1993; PCT 92/05254 published April 2, 1992; Hallbook et al. , Neuron, 6: 845-858

[1991]), and NT-5. These polypeptides have been shown to function in the growth, survival, and / or differentiation of various types of nerve cells. BDNF, NT-3, NGF, and NT-4 share a significant 'homology in the amino acid sequence. BDNF and NGF are approximately 55% homologous at the amino acid level, and NT-3 is approximately 58% homologous to BDNF and approximately 57% homologous to NGF (Narhi et al., J. Biol. Chem., 268: 13309- 17

[1993]). NT-4 is approximately 46%, 55% and 52% homologous with mature NGF, BDNF and NT-3, respectively at the amino acid sequence level (PCT 92/05254, published on April 2, 1992; see also Hallbook et al., Neuron, 6: 845-858

[1991]). The NGF polypeptides contain various cysteine residues, and the amino acid sequence in the region of these cysteines is relatively conserved (Narhi et al., Supra). Due to their respective roles in the maintenance of the nervous system, it was anticipated that NGF, NT-3, NT-4 and BDNF, when prepared in biologically active form, will be useful as therapeutic agents in the treatment of various diseases or conditions of the nervous system. Conditions such as damage to the nervous system (via traumas, surgery, infection, exposure to toxins and / or malnutrition), various neuropathies, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amilotropic lateral sclerosis, Huntington's chorea, extrafamilial strains , and similar.

Related Technique US Patent No. 5,235,043 issued August 10, 1993 was intended to describe the processes for producing members of the mature human NGF (NT-3) / BDNF family of neurotrophic proteins that are biologically active in their entirety. The intended polypeptides can be secreted from the host cells using a signaling peptide such as the OmpA signal peptide. The North American Patent No. 4, 757,013 issued July 12, 1988 describes a cloning plasmid useful for the secretion of polypeptides in bacterial hosts. In addition to other nucleic acid sequences, the plasmid optionally contains a DNA fragment encoding the E. coli OmpA protein signaling peptide. It was reported that the plasmid provides efficient secretion through the cytoplasmic membrane. U.S. Patent No. 4,338,397 issued July 6, 1982 describes a method for the secretion of a polypeptide produced in a bacterial host cell. The reported method allows the production of proteins free of chemical substituents such as "f-met". Tanji et al (J. Bacteriol., 173: 1997-2005

[1991]) describe certain substitutions or deletions of amino acids in the amino acid sequence of the OmpA signal peptide. Golstein et al (J. Bacteriol., 172: 1225-1231

[1990]) describe mutants of the OmpA signaling peptide with altered levels of hydrophobicity. Reportant mutants have different abilities to act as signaling peptides for two proteins through the E. Coli cell membrane. Lenhardt et al (J. Biol. Chem., 263: 10300-03

[1988]) describe mutants of the OmpA signaling peptide, in which the net charge of the amino terminal end was reported altered. Lenhardt et al (J. Biol. Chem., 262: 1716-19

[1987]) describe the production of OmpA signaling peptide mutants. The mutants had a shortened hydrophobic region, and their ability to act as secretion sequences for certain proteins was reported to be altered. In view of the problems that have been encountered in the preparation of large quantities of polypeptides using recombinant DNA technology, there is a need for the art of providing methods for increasing the yields of such polypeptides produced for recombinant DNA technology. In addition, there is a need in the art to provide recombinant polypeptides in the "non-Met" form. Accordingly, an object of the present invention is to provide a signaling peptide, and the nucleic acid sequences encoding the signaling peptide, which serves to increase the efficiency of the secretion of the NGF polypeptide produced in a prokaryotic host cell. Yet another object is to provide a method for preparing NGF polypeptide in the non-Met form. The other objects will be apparent to one skilled in the art.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, in one aspect, the invention provides a signaling peptide comprising the sequence MKKRARAIAIAVALAGFATVAHA (SEQ ID NO: 1). In another aspect, the signaling peptide may further comprise at its terminal carboxyl the BDNF, _NGF, NT-3, NT-4, or NT-5 polypeptide. In another aspect, the invention provides a nucleic acid comprising a sequence as set forth in SEQ ID NO: 2. In another aspect, the invention provides a nucleic acid as set forth in SEQ ID NO: 3. In another aspect, the invention provides a nucleic acid as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 further comprising at its 3 'end a nucleic acid encoding a NGF polypeptide without an amino terminal meth.

In yet another aspect, the invention provides a vector comprising the nucleic acid as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 ligated at its 3 'end to a nucleic acid encoding a NGF polypeptide without an amino terminal . Optionally, the vectors can be pCFM1656 / BDNFopt3 or pCFM656 / NT-3opt3. In another aspect, the invention provides a prokaryotic host cell in which the vector has been inserted. In another aspect, the invention provides a method for producing a non-Met form of an NGF polypeptide comprising culturing a prokaryotic host cell in which a vector comprising the nucleic acid as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 bound at its 3 'end to a nucleic acid encoding a NGF polypeptide without an amino terminal meth has been inserted, and isolate the secreted NGF polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the amino acid sequence of a synthetic signaling peptide, RH, useful for increasing the secretion of certain E. coli cell polypeptides. (SEQ ID NO: l).

Figure 2 depicts a degenerate nucleotide sequence encoding the RH signaling peptide. "R" represents A or G; "W" represents A or T / U; "Y" represents C or T / U; and "D" represents A, G, or T / U (SEQ ID N0: 2). The sequence was designed for the preferential use of the codon to prokaryotic cells. Figure 3 describes a nucleotide sequence, RHll, which codes for the RH signaling peptide (SEQ ID NO: 3). Figures 4A and 4B describe the sequences of Synthetic DNA that code for a sequence of amino acids of human BDNF that is useful for the preparation and expression of BDNF in a prokaryotic host cell. 4A (SEQ ID NO: 4) describes the sequence with a start codon of Met and 4B (SEQ ID NO: 22) describes the same sequence minus the start codon of Met ATG. Figures 5A and 5B describe the synthetic DNA sequences encoding an amino acid sequence of human NT-3 that is useful for the preparation and expression of NT-3 in a prokaryotic host cell. The 5A (SEQ ID NO: 5) describes the sequence with a start codon of Met and the 5B (SEQ ID NO: 23) describes the sequence minus the start codon of Met ATG. Figure 6 is a schematic diagram of the strategy used to prepare a synthetic BDNF nucleic acid sequence linked at its 5 'end to a nucleic acid sequence encoding the RH signaling peptide. "SP" indicates the peptide signaling sequence; the selected restriction enzymes are shown. The relative size of each nucleic acid sequence is not to scale.

DETAILED DESCRIPTION OF THE INVENTION This invention was based on the unexpected discovery that certain synthetic signal peptide sequences serve to increase the amount of NGF polypeptides secreted from some prokaryotic host cells.

Preparation of the Invention The present invention contemplates the use of the RH signaling peptide to increase the expression and secretion of the polypeptides of the nerve growth factor (NGF) family synthesized and expressed in prokaryotic host cells. These polypeptides will be referred to herein as "NGF polypeptides".

In addition, the invention provides the means for introducing an NGF polypeptide lacking a methionine to its amino terminus. Such NGF polypeptides are called NGF "without Met" polypeptides herein. 1. Preparation of Rh Nucleic Acid Sequences Included within the scope of this invention are all possible nucleic acid sequences encoding the RH signaling peptide. Partially degenerate codon nucleic acids (ie, designed for the preferential use of the codon in prokaryotic cells) for the RH are set forth in Figure 2. One of the preferred nucleic acids encoding the RH is the RHll, the sequence of which is set forth in Figure 3. The nucleic acids encoding the RH can be readily prepared using methods well known in the art, such as those set forth by Engels et al. (Agew. Chem. Intl. Ed., 28: 716-734

[1989]). A preferred method for synthesis is synthesis supported by polymers using the chemical method of the standard phosphoramidite. 2. Preparation of the DNA encoding the NGF Polypeptides Included within this invention is a method for preparing a NGF polypeptide from a mammalian source, including without limitation, human, bovine, and porcine. A preferred source is the human. The NGF polypeptides included within the scope of this invention are, without limitation, BDNF (neurotrophic factor derived from the brain), NT-3 (neurotrophin 3 also known as NGF 3), NGF (nerve growth factorj), NT-4 (neurotrophin 4), NT-5 (neurotrophin 5), and other members of this family related to their significant amino acid or nucleic acid sequence homology, such as that exhibited by members of this family known up to now. which codes for the NGF polypeptides contemplated herein can be isolated and obtained in a suitable amount using one or more of the methods that are well known in the art.These methods and other useful for isolating such DNA are disclosed, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[1989]) and Berger and Kimmel (? Ies? In in Enzymology: Guide to Molecular Cloning Techniques, vol. 152, Academic P Ress, Inc., San Diego, CA

[1987]). The preferred nucleic acid sequences for BDNF are those set forth in Figures 4A and 4B. The preferred nucleic acid sequences for NT-3 are those set forth in Figures 5A and 5B. Where the amino acid sequence of the NGF polypeptide is known, a probable and functional nucleic acid encoding the polypeptide can be inferred using the known and / or preferred codons for each residual amino acid. Where the nucleic acid sequence of the NGF polypeptide is completely known, this sequence can be synthesized, in whole or in part, using chemical synthesis methods such as those described in Engels et al. (Angew. Chem. Int. Ed. Engl., 28: 716-734

[1989]). These methods include, inter alia, nucleic acid synthesis methods with phosphotriester, phosphoramidite and H-phosphonate. Typically, the DNA encoding the polypeptide will be several hundred base pairs (bp) or nucleotides in length. Nucleic acids greater than about 100 nucleotides in length can be synthesized as several fragments, each fragment is up to about 100 nucleotides in length. The fragments can then be ligated, as described below, to form a full-length nucleic acid encoding the NGF polypeptide. Alternatively, the nucleic acid encoding the NGF polypeptide can be obtained by selecting an appropriate cDNA (i.e., prepared from a tissue source believed to express the polypeptide) or genomic library using one or more nucleic acid probes (oligonucleotides , CDNA or genomic DNA fragments with an acceptable level of homology with the nucleic acid to be cloned, and the like) will hybridize selectively with the desired nucleic acid. Another suitable method for obtaining a nucleic acid encoding the NGF polypeptide is the polymerase chain reaction (PCR). However, the successful use of this method requires that sufficient information about the nucleic acid sequence encoding the NGF polypeptide be available to design suitable oligonucleotide primers useful for the amplification of the nucleic acid sequence. Where the method of choice for preparing the nucleic acid encoding the NGF polypeptide requires the use of primers or oligonucleotide probes (for example PCR, cDNA or selection of the genomic library), the oligonucleotide sequences selected as probes or primers should be of the appropriate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that will occur during the selection of the library or PCR. The actual sequence of the probes or primers is usually based on the sequences or regions conserved or highly homologous thereto or a similar gene of another organism. Optionally, the probes or primers may be degenerate. In the case where only the amino acid sequence of the NGF polypeptide is known, one can infer probable or functional nucleic acid encoding the polypeptide sequence using known and preferred codons for each residual amino acid. This sequence can then be synthesized chemically using the methods described above. This invention includes the preparation of mutant sequences for the NGF polypeptide. A mutant sequence as used herein contains one or more substitutions, deletions and / or nucleotide insertions compared to the wild-type sequence. Substitution, deletion and / or nucleotide insertion can result in an NGF polypeptide that is different in its amino acid sequence from the wild-type amino acid sequence. The preparation of such mutants is well known in the art, and is described for example in Wells et al. (Gene, 34: 315

[1985]), and in Sambrook et al. supra. 3. Preparation of NGF Polypeptide Precursor A nucleic acid encoding the NGF polypeptide precursor (ie, the form containing the signaling peptide sequence) can be prepared using any of a variety of methods. In one method, a nucleic acid encoding the RH can be directly linked to the nucleic acid encoding the NGF polypeptide, provided that the nucleic acid encoding the NGF polypeptide does not contain the codon for the amino terminal methionine. The ligation of the two nucleic acids can be performed by blunt-end ligation or by designing a suitable restriction endonuclease site at the 3 'end of the RH nucleic acid, using the enzyme ligase and following the manufacturer's protocol, or by the methods well known in the art such as those described by Sambrook et al. supra. Alternatively, the nucleic acid encoding the RH can be connected to the nucleic acid sequence of the NGF polypeptide using the PCR. Here, the RH nucleic acid containing the entire RH sequence, plus 15-21 additional nucleotides at its 3 'end containing the first 5 to 7 codons (ie, 5') of the nucleic acid sequence coding for an NGF polypeptide excluding the Met codon (usually ATG) is used as the first PCR primer. The NGF RH / 5 'nucleic acid primer is synthesized using well known methods such as the chemical method of the standard phosphoramidite. The second PCR primer can be a nucleic acid that is complementary to the last 6-8 codons (18-24 rn-cleotides) of the NGF nucleic acid ie the codons at the 3 'end of this nucleic acid. In this way, using the PCR, a nucleic acid sequence consisting of the RH linked to the full-length nucleic acid encoding the NGF polypeptide minus the codon of the amino-terminal Met of the nucleic acid encoding this NGF polypeptide. Another useful method for preparing the nucleic acid encoding the NGF precursor polypeptide also uses PCR. Here, the full length nucleic acid encoding the NGF polypeptide (which may contain the amino terminal Met codon) is first inserted into a cloning or expression vector. The PCR is then used to prepare a nucleic acid containing an RH sequence linked to the full-length nucleic acid encoding the NGF polypeptide. The primers of the PCR are similar to those described above. A primer used for PCR is complementary to the vector sequence located 3 'to the region of the nucleic acid encoding the NGF polypeptide. The other primer contains the 5 'to 3' order, a portion (approximately 12-25 nucleotides) of the vector sequence that is immediately 5 'to the sequence encoding the NGF; a full-length nucleotide sequence coding for the RH signaling peptide, and the nucleotide sequence coding for the first 5-8 amino acids excluding the amino-terminal Met of the NGF polypeptide. When this primer is hybridized to the vector containing the nucleic acid insert encoding the NGF polypeptide, the portion of the vector sequence and the portion of the sequence encoding the NGF polypeptide can hybridize to the primer while the of the RH sequence no. The portion of the RH can form a circular structure. The other PCR primer for use in this method may be a nucleic acid sequence that is complementary to the last 6-8 codons (nucleotides 18-24) of the nucleic acid encoding the NGF polypeptide, or to the portion of the sequence of the 3 'vector to the NGF nucleic acid sequence.

During PCR, the amplified nucleic acid product will contain, 5 'to 3' a portion of the 5 'vector sequence, a sequence of the RH signal peptide, the full-length nucleic acid encoding the NGF polypeptide, minus the amino terminal Met codon, and a portion of the 3 'vector sequence. After PCR, the PCR product can be cut with suitable restriction enzymes to generate a nucleic acid encoding a precursor of a NGF polypeptide, which produces a NGF polypeptide without an amino-terminal meth after the signaling peptide of RH breaks during secretion. 4. Preparation / Selection of a vector.

Any expression vector that is functional in the selected prokaryotic host cell can be used as long as the vector contains all the components or elements of the nucleic acid necessary to ensure expression of the NGF precursor polypeptide. Typically, the vector will contain a promoter, an origin of the duplication element, a transcriptional termination element, an element of ribosomal binding sites, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

A. Promoter element.

The promoter can be homologous (ie, from the same species and / or prokaryotic strain as the host cell), heterologous (ie, from a different source to that of the prokaryotic host cell species or strain), or synthetic. As such, the source of the promoter can be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant as long as the promoter is functional and can be regulated by the host cell. The most preferred promoters of this invention are inducible promoters such as those originating in the bacteriophage lambda, i.e., lambda promoters such as the PR or PL promoters, the T5 promoter or the T7 promoter; bacterial promoters such as lac, tac (a composition of the trp and lac promoters), trp, and tna. A more preferred promoter is the promoter PL- The nucleic acid sequences of the promoter useful in this invention can be obtained by any of several methods well known in the art. Typically the promoters useful herein will have to be previously identified by mapping and / or by restriction endonuclease digestion and can thus be isolated from the appropriate tissue source using the appropriate restriction endonucleases. In some cases, the promoter may have been sequenced. For those promoters whose DNA sequence is known, the promoter can be synthesized using the methods described above for the synthesis or cloning of the nucleic acid. Where all or only portions of the promoter sequence are known, the promoter can be obtained using the PCR and / or by selecting a genomic library with the fragments of the oligonucleotide sequence and / or the suitable promoter from the same or other species. Where the sequence of the promoter is not known, a fragment of the DNA containing the promoter of a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes, can be isolated. Isolation can be carried out by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the appropriate DNA fragment. After digestion, the desired fragment can be isolated by purification on agarose gel, Qiagen® column or other methods known to those skilled in the art. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one skilled in the art.

B. Origin of the Duplication Element This component is typically a part of commercially purchased prokaryotic expression vectors, and aids in the amplification of the vector in the host cell. The amplification of the vector to a certain number of copies may, in some cases, be important for the optimal expression of the NGF polypeptide. If the vector of choice does not contain an origin of the duplication site, one can be synthesized chemically based on a known sequence, and ligated into the vector.

C. Transcription of the Completion Element This element is typically located 3 'to the end of the sequence encoding the NGF polypeptide and serves to terminate transcription of the NGF polypeptide. Usually, the transcription termination element in the prokaryotic cells is a GC-rich fragment followed by a poly T sequence. Although the element is easily cloned from a library or even commercially purchased as part of a vector, it can also be be easily synthesized using the methods for nucleic acid synthesis such as those described above.

Element of Selectable Markers Selectable marker genes encode the proteins necessary for the survival and growth of a host cell that grows in a selective culture medium. Selection marker genes typically code for proteins that, (a) confer resistance to antibiotics or other toxins, eg, ampicillin, tetracycline, or kanamycin by prokaryotic host cells, (b) complement auxotrophic cell deficiencies; or (c) provide critical nutrients not available from the complex medium. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.

E. Element of the Ribosomal Union Site This element, commonly called the Shine-Dalgarno sequence, is necessary for the initiation of mRNA translation. The element is typically located 3 'to the promoter and 5' to the coding sequence of the polypeptide to be synthesized. The sequence of Shine * Dalgarno is varied but is typically a polypurine (ie, it has a high content of A-G). Many Shine-Delgarno sequences have been identified, each of which can be easily synthesized using the methods discussed above. All of the elements discussed above, as well as others useful in this invention, are well known to those skilled in the art and are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[1989]) and Berger et al. , eds. . { Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, CA

[1987]).

F. Construction of Vectors Most of the vectors useful in the practice of this invention are any expression vector that is compatible with the selected prokaryotic host cell. In certain cases, some of the different elements of the vectors listed above may already be present in commercially available vectors such as vectors pUC18, pUC19, pGEM vectors (Promega Corp, Madison, WI), pBluescript® vectors such as pBIISK +/- (Stratagene Corp., La Jolla, CA), and the like, all of which are suitable for prokaryotic host cells, and suitable for use in the practice of this invention. Where one or more of the elements are not already present in the vector to be used, they can be obtained and ligated individually to the vector. The methods used to obtain each of the elements are well known to those skilled in the art and are comparable to the methods set forth above (ie, DNA synthesis, library selection, and the like). Preferred vectors of this invention are vectors pCFM1656 (deposited on February 24, 1993 under the Budapest Treaty with the American Type Culture Collection, 12301 Parkla n Drive, Rockville, MD 20852, as accession number 69576), pGEM, the vectors pBluescript®, pUC18, and pUC19. The plasmid pCFM1656 requires a culture temperature of about 30 ° C to about 42 ° C to obtain an increase in the copy number of the plasmid; increasing the temperature above about 42 ° C inactivates the repressor element CI857 of the PL promoter. The final vector used for the practice of this invention is typically consumed from a starting or starting vector such as a commercially available vector. This vector may or may not contain some of the elements to be included in the complete vector. If none of the elements are present in the starting vector, each element can be ligated individually to the vector by cutting the vector with the appropriate restriction endonuclease, so that the ends of the element to be ligated and the ends of the vector are compatible for ligation. In some cases, it may be necessary to "cut" the ends to be ligated to obtain a satisfactory ligation. The cut is made by first filling the "sticky ends" using the Klenow DNA polymerase or the T4 DNA polymerase in the presence of the four nucleotides. This method is well known in the art and is described, for example, in Sambrook et al. , supra.

Alternatively, two or more of the elements to be inserted in the vector can be linked first (if they are to be placed adjacent to each other) and then ligated into the vector. Another method for constructing the vector is to conduct all the ligations of the different elements simultaneously in a reaction mixture. Here, many non-sense or non-functional vectors will be generated due to the ligation or inappropriate insertion of the elements, however, the functional vector can be identified and selected by restriction endonuclease digestion. After the vector has been constructed and the NGF nucleic acid has been inserted into the appropriate site of the vector, the entire vector can be inserted into the prokaryotic host cell for amplification and expression of the NGF polypeptide. The prokaryotic cells typically used are any strain of E. coli that are compatible with the promoter on the vector, so that the promoter is functional in the E. coli cell. Preferably, the selected strain should provide the regulatory elements that control the promoter, so that expression of the NGF polypeptide is induced appropriately. Such strains as strains FM-5 of E. coli (deposited on May 19, 1989 under the Budapest Treaty with the ATCC as accession number 53911), DH5-a, JM-101, and simlares are suitable. In addition, other gram-negative bacteria such as salmonella, as well as gram-positive bacteria such as bacillus and other prokaryotes may be suitable as host cells. The insertion (also known as transformation or "transfection") of the vector into the selected host cell can be effected using methods such as the calcium chloride method, electroporation, microinjection, lipofection or DEAE dextran. The method selected separately will be a function of the type of host cell to be used. Those methods and other suitable methods are well known to those skilled in the art, and are discussed, for example, in Sambrook et al. , supra. Host cells that contain the vector (ie, transformed) can be cultured using standard means well known to those skilled in the art. The media will usually contain all the nutrients necessary for the growth and survival of the cells. Suitable means for culturing E. coli cells are for example, Luria Broth (LB) and / or Terry Broth (TB). Typically, an antibiotic or other compound useful for the selective growth of transformed cells is added alone, as a supplement to the medium. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell It was transformed. For example, where the selectable marker element is resistance to kanamycin, the compound added to the culture medium should be kanamycin. 5. Evaluation of the Secretion The amount of NGF polypeptide secreted from the host cell and thus converted to the mature, non-Met form can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, CLAP separation, immunoprecipitation, and / or activity assays. To conduct some of the assays listed above, it may be necessary to first manipulate the culture material of the host cell to extract the polypeptide. Typically, the amount of processed, or mature, polypeptide secreted by the host cell will be compared to the total amount of the expressed polypeptide (mature precursor polypeptide). Extracts of host cells can be prepared using standard methods of the art such as those set forth by Culi et al (Meth. Enz., 182: 147-153

[1990]). If the host cells used are gram positive or other bacteria prokaryotes that have only a single cytoplasmic membrane, the secreted polypeptide can be found in the cell culture medium. Where the host cells are gram negative bacteria such as E. coli or other prokaryotes with two membranes (inner and outer membranes), the majority of secreted polypeptide can be found in the polyplastic space, ie the space between the inner and outer membranes . The collection of the unprocessed form of the NGF polypeptide (ie, the precursor form) will typically require extraction of the polypeptide from the host cell. For most assays such as gel electrophoresis, Western blotting and spot blots, the NGF precursor polypeptide does not need to be purified. Instead, host cells that contain it can simply be harvested by precipitating them into a pellet in a centrifuge, and then being lysed using standard methods. The cell debris (membranes, cell wall material and the like) can be separated by precipitating them into a pellet in a centrifuge ", and the soluble fraction can then be swallowed directly onto a gel or dot blotter for further analysis. The methods used to collect the processed or secreted form of the NGF polypeptide will depend on whether the polypeptide is located in the periplasm (gram negative bacteria) or in the culture medium (gram positive bacteria and other prokaryotes). find it in the culture medium, an aliquot of the culture medium can be used directly for gel electrophoresis analysis of spot spots, and / or immunoprecipitation.The NGF polypeptide is expected to be mainly in the periplasmic space, the periplasmic content, including the inclusion bodies if the processed polypeptide has formed such complexes, can in being extracted from the host cell using any standard technique known to those skilled in the art. For example, the host cells can be lysed to release the periplasmic contents by a French press, homogenization, and / or sonication. The homogenate can then be centrifuged. If the NGF polypeptide has formed inclusion bodies in the periplasm, the inclusion bodies can often bind to the inner and / or outer cell membranes and "thus will be found mainly in the pellet material after centrifugation. The pellet material can then be treated with a chaotropic agent such as guanidine or urea to release, break and solubilize the inclusion bodies.The NGF polypeptide now in its soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or If it is desired to isolate the NGF polypeptide, the isolation can be carried out using standard methods, such as those set forth below and in Marston et al., (Meth. Enz., 182: 264-275

[1990]). If the NGF polypeptide did not form inclusion bodies to a significant extent in the periplasm of the host cell, the NGF polypeptide will mainly be found in the supernatant After centrifugation of the cell homogenate, and the NGF polypeptide can be isolated from the supernatant using methods such as those discussed below. In those situations where it is preferable to partially or completely isolate the precursor and / or mature forms of the NGF polypeptide, purification can be carried out using standard methods well known to those skilled in the art. Such methods include, without limitation, separation by electrophoresis followed by electroelination, various types of chromatography (immunoaffinity, molecular sieve and / or ion exchange), and / or high pressure liquid chromatography. In some cases, it may be preferable to use more of those methods to complete the purification. The invention can be more easily understood by reference to the following examples. These examples, should not be construed in any way as limiting the scope of the invention.

EXAMPLES Example 1: Secretion of BDNF A. Preparation of DNA Constructs The human brain derived neurotrophic factor (BDNF) was prepared for expression in E. coli cells as follows. First, a synthetic BDNF gel was designed to provide improved expression in E. coli cells. This gene tubes several altered codons at one or more bases compared to the nucleic acid sequence for human BDNF found in nature. The sequence of the synthetic gene, called BDNFopt3, is set forth in Figure 4. The gene codes for a methionine (ATG) at the 5 'end of the gene. BDNFopt3 was prepared with polymer-supported synthesis using the standard phosphoramidite chemical methods. Due to the length of BDNFopt3, the gene was synthesized as four separate segments: segment 1 is 104 bases and contains some 5 'untranslated sequences corresponding to the vector sequence, a Xbal restriction site, an ATG start codon , and the first 76 bases of the BDNFopt3 nucleic acid sequence; segment 2 contains the following 117 bases of BDNFopt3? segment 3 contains the following 107 bases of BDNFopt3; and segment 4 contains the remaining 57 bases of BDNFopt3 together with the stop codon or high TAA, a sequence of the BamHI restriction site, and 5 additional nucleotides. The segments were ligated using standard ligation protocols. Prior to ligation, three oligonucleotides were hybridized to the BDNFopt3 gene fragments to ensure four segments of the gene were ligated in the proper order. Each of the three oligonucleotides used encompassed one of the junctions of the BDNFopt3 gene fragment. The nucleic acid sequence of each of these oligonucleotides is set out below: ACTGCGGTTTTCTTATCAGC (SEQ ID NO: 6) CAGCCTTCTTTAGTGTAACC (SEQ ID NO: 7) GGATGAAGCGCCAGCCGATA (SEQ ID NO: 8) After ligation of the segments, a small portion of the ligation mixture was used with the gene Full-length single-stranded BDNFopt3 as a standard for PCR to amplify this gene. The primers used for the PCR amplification were: TTGATTCTAGAAGGAGGAA (SEQ ID NO: 9) TCCGCGGATCCTTAGCGGCC (SEQ ID NO: 10) Twenty-five cycles of PCR were conducted, using the following conditions: denaturation at 94 ° C for one minute; the annealing was at 55 ° C for one minute; and the extension was at 72 ° C for two minutes. The amplified fragment was purified from an agarose gel using the GENECLEAN II kit (Bio 101, Inc., La Jolla, CA), and digested with the restriction enzymes Xbal and BamHI. The fragment was then inserted into vector pCFM1656 (ATCC accession number 69576) previously cut with Xbal and BamHI. This vector containing the full-length BDNFopt3 gene, called pCFM1656 / BDNFopt3, was used as a standard to prepare the secretable form of the BDNFopt3 gene (ie, containing a signaling sequence). The cloning strategy used to prepare the secretable form of BDNFopt3 is described in Example 6. The deneged oligonucleotide sequences encoding the signaling peptide sequence set forth in Figure 2 were prepared using the supported or supported synthesis by polymer and the chemical method of the standard phosphoramidite Each sequence had at its 5 'end approximately 24 bases of the vector sequence pCFM1656 localized 5' to the sequence of the BDNFopt3 gene in pCFM1656 / BDNFopt3 and at its 3 'end approximately 18 bases coding for the first 6 amino acids of BDNFopt3 (excluding aminoterminal methionine, which was omitted to prepare the non-Met form of secreted BDNFopt3) • To prepare the secreted form of BDNFopt3, the oligonucleotides were annealed with the degenerate RH signaling peptide codons, and a second oligonucleotide that is complementary to the vector sequence downstream of the mo 3 'of the BDNFopt3 gene (shown below as SEQ ID NO: ll), for pCFM1656 / BDNFopt3. GTTGCTGCGGATTCTCACCAA (SEQ ID NO: 11) The polymerase chain reaction (PCR) was then used to synthesize a gene coding for the entire sequence of BDNFopt3 attached at its 5 'end to a nucleic acid encoding the peptide sequence of RH signaling. PCR was conducted using the reagents and the DNA polymerase tag obtained from Boehringer Mannheim Biochemicals, Inc. Twenty-five PCR cycles were conducted as follows: the denaturation was at 94 ° C for 1 minute, the annealing was at 50 ° C for 1 minute. minute, and the extension was at 72 ° C for 2 minutes. After PCR, the PCR product (approximately 574 base pairs encoding BDNFopt3 plus the RH signaling peptide) was run on a 0.8% agarose gel, and the 574 base pair fragrance was cut of the gel and purified using the GENECLEAN II equipment (Bio 101, Inc.) and following the manufacturer's instructions. The purified fragment was digested with XbaI and XhoI to generate a DNA fragment of approximately 200 base pairs which codes for the RH signaling peptide and approximately one third of the 5 'of the BDNFopt3 gene. The vector pCFM1656 / BDNFopt3 was digested with XbaI and XhoI, the small fragment coding for the 5 'end of the RH / BDNFopt3 was removed, at the frequency of the RH / BDNFopt3 of 200 base pairs it was ligated to this cut vector. The ligation was overnight at about 16 ° C using ligase buffer and the enzyme obtained from Boehringer Mannheim, Inc., and carrying out the reaction according to the manufacturer's instructions. After ligation, the plasmids were inserted (transformed) into competent E. Coli K-12 cells of strain FM-5 (ATCC accession number 53911). Insertion of the plasmid was carried out using the standard calcium chloride method as set forth in Sambrook et al. , supra. Transformed cells were grown overnight at about 30 ° C on standard Luria broth agar plates ("LB") containing kanamycin at approximately 50 μg / ml. After culturing, 16 individual colonies were selected, inoculated into the LB containing kanamycin at about 50 μg / ml, and cultured overnight at about 30 ° C on a shaker. After culturing, the cells decreased to approximately 1:10 in standard Termite broth ("TB"; Sambrook et al., Supra) containing kanamycin, and allowed to grow at approximately 30 ° C to a density of approximately 0.5-0.8 OD600 , at which time the temperature was increased to approximately 42 ° C to induce the expression and secretion of BDNFopt3. After approximately 4 hours, approximately 1 mg of moist cell mass was centrifuged and then boiled for approximately 10 minutes in approximately 100 μl of standard SDS-PAGE Tris-glycine denaturation / reduction buffer (62.5 mM Tris-HCl, pH 6.8, 2% of SDS, bromophenol blue 0.0025%, glycerol 10%, ß-mercapto-ethanol 2.5%). Approximately 10 μl of this mixture was then run on a standard SDS polyacrylamide gel (approximately 18 percent acrylamide). The gel was run at a constant voltage of about 130 volts for about 2.5 hours. The gels, sample buffer and run buffer were all obtained from NOVEX, Inc. (San Diego, CA) and were used according to the manufacturer's instructions. As a control system, a nucleic acid encoding the signaling peptide for the OmpA was linked to the BDNFopt3 gene using the same procedures set forth above for the preparation of the RH / BDNFopt3 nucleic acid. The oligonucleotide sequence used to generate the OmpA signaling peptide was: ATTCTAGAAGGAGGAATAACATATGAAAAAGACAGCTATCGCGATT GCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCCACTCTG ACCCGGCTCGT (SEQ ID NO: 12).

As for the oligonucleotides of the RH, the oligonucleotide OmpA had at its 5 'end about 24 bases' of the vector sequence pCFM1656 and at its 3' end about 18 bases coding for the first 6 amino acids of the BDNFopt3 (excluding the amino terminal Met ).

B. Analysis of the Nucleic Acid Sequence of Signaling Peptide The efficiency of the different nucleotide sequences encoding the RH signaling peptide was analyzed as the percentage of the amount of processed BDNFopt3 (cut signaling peptide of the polypeptide) compared to the total BDNFopt3 protein produced (processed plus unprocessed). To compare the two forms of BDNFopt3, the SDS-gel was stained with Commassie blue, and scanned after staining to determine the relative amount of each form. The molecular weight of the crude precursor polypeptide of BDNFopt3 is about 15.8 kD, while the molecular weight of the processed form of BDNFopt3 is about 13.5 kD. Thus, the two forms of BDNFopt3 show distinctly different bands on the denaturing polyacrylamide gel with SDS.

For a number of RH / BDNFopt3 polypeptides, both bands (ie, processed and unprocessed BDNFopt3) were visually evident both for Commassie blue gel staining and for Western blotting of the gel. BDNFopt3 on Western blotting with a polyclonal anti-BDNF antibody. A nucleic acid sequence of RH, RHll, which appeared by visual inspection has more processed BDNFopt3 compared to the other RH nucleic acid sequences such as RH12, and to the nucleic acid sequence of OmpA. The gel was explored using an Ultrascan XL (Pharmacia LKB) to determine the relative amounts of Processed and unprocessed BDNF for the different nucleic acid sequences of the different RH signaling peptide and the nucleic acid sequence of the OmpA signaling peptide. The results of the screening for the nucleic acid sequences of RHll, RH12, and OmpA are shown in Table 1 below. The results were reported as the area under the curve (Absorbance x width in millimeters of the band of protein on gel) obtained from the gel scan.

Table 1 OmpA RHll RH12 Unprocessed 0.64 0.07 1.61 Processed 0.34 0.65 0.73 Percent processed 34.7 90.3 31.2 As is evident, the use of the nucleic acid sequence of the RHII signaling peptide resulted in a more effective processing of BDNFopt3 compared to the use of the OmpA or RH12 sequences.

Example 2: Secretion of NT-3 A. Preparation of DNA Constructs A synthetic gene encoding human NT-3 was prepared for expression in E. Coli cells. The gene was designed to improve expression in those cells by using the preferred bacterial codons. The sequence of this synthetic gene is set forth in Figure 5. The synthetic gene, called NT-3opt3, was prepared by polymer-supported synthesis using the chemical methods of the standard phosphoramidite. Due to the length of NT-3opt3, it was prepared as four separate nucleic acid fragments whose lengths ranged from 94 a. 104 nucleotides. The fragment coding for the 5 'portion of the NT-3opt3 gene had some 5' non-coding sequence to the ATG start codon to provide an XibaJ site. The fragrance coding for the 3 'portion of the NT-3opt3 gene had some non-coding sequence at the 3' end, which provides a BamHI restriction site. After the synthesis, the fragments were ligated using oligonucleotide binding nucleic acids whose sequences are complementary to the regions around each binding ligation. Annealing and ligation were performed using the standard protocols. The oligonucleotide sequences used as binding sequences are set forth below: AGCGGAGGATTTGTCGGTAA (SEQ ID NO: 13) CTTTGCAACGGGTTTCGTAG (SEQ ID NO: 14) GTTTTCGGAGGTCAGAGCAC (SEQ ID NO: 15) A small fraction of the mixture bound to the full-length single-stranded NT-3opt3 gene was used as a standard for PCR to amplify the NT-3opt3 gene.

The following two oligonucleotides were used as primers for this PCR: TTGATTCTAGAAGGAGGAAT (SEQ ID NO: 16) TCCGCGGATCCTTAGGTACG (SEQ ID NO: 17) Twenty-five cycles of PCR were conducted as follows: the denaturation was at 94 ° C for 1 minute; the annealing was at 55 ° C for 1 minute; and the extension was at 72 ° C for 2 minutes. The amplified fragment was purified from agarose gel using the GENECLEAN II kit (Bio 101), digested with Xbal and BamHI, and inserted into the vector pCFM1656 previously cut with Xbal and BamHI. This plasmid containing the NT-3opt3 gene was used to prepare the NT-3opt3 gene containing a nucleic acid encoding the peptide sequence of RH signaling. The strategy used was comparable to that set forth for BDNFopt3 in Example 1 and described in Figure 6. The partially degenerate oligonucleotide sequences encoding the sequence of the RH signaling peptide as set forth in Figure 2 were synthesized using the methods of DNA synthesis with phosphoramidite. Each sequence had at its 5 'end approximately 24 bases of the vector sequence pCFM1656. Each sequence had at its 3 'end 18 base pairs that code for the first 6 amino acids of NT-3opt3 (excluding the first residual amino acid methionine, which was omitted to prepare a form of the NT-3opt3 secreted without Met). To prepare a secreted form of NT-3opt3, the oligonucleotide sequences containing the codons of the RH signaling peptide were annealed to the vector pCFM1656 / NT-3opt3 by PCR. A second oligonucleotide sequence that is complementary to the 3 'end of the vector sequence downstream of in NT-3opt3 to pCFM1656 / NT-3opt3 was simultaneously annealed. Next, the polymerase chain reaction (PCR) was used to synthesize a gene coding for the entire NT-3opt3 sequence bound at its 5 'end to a nucleic acid encoding the sequence of the RH signaling peptide. PCR was conducted using the reagents and DNA polymerase tag from Boehringer Mannheim Biochemicals, Inc. Twenty-five PCR cycles were conducted as follows: the denaturation was at 94 ° C for 1 minute. The annealing was at 50 ° C for 1 minute, and the extension was at 72 ° C for 2 minutes. After PCR, the PCR product (approximately 574 base pairs that code for the NT-3opt3 and the signaling peptide RH) was run on a 0.8% agarose gel, and the RH / NT-3opt3 nucleotide fragment of approximately 574 was cut out of the gel and purified using the GENECLEAN II kit (Bio 101, Inc. ) following the manufacturer's instructions. The purified fragment was digested with XbaJ and HindIII to generate an approximately 371 bp DNA fragment encoding the RH signal peptide and the 5 'portion of the NT-3 gene. The vector pCFM1656 / NT-3opt3 was digested with Xbal and HindIII, the fragment coding for the 5 'portion of the NT-3opt3 was removed and replaced by ligation with the DNA sequence of the RH / NT-3opt3 of approximately 371 base pairs ( produced by PCR). The ligation was overnight at 16 ° C using ligase buffer and the enzyme obtained from Boehringer Mannheim, Inc., and following the manufacturer's instructions. After ligation, the plasmids were inserted (transformed) into competent E. Coli K-12 strain FM-5 (access code 53911, A.T.C.C.). Insertion of the plasmid into the E. coli cells was performed using the standard calcium chloride method as set forth in Sambrook et al. , supra. Transformed cells were grown overnight at about 30 ° C on standard Luria broth agar plates ("LB") containing kanamycin at approximately 50 μg / ml. After culturing, 13 individual colonies were selected, inoculated in the LB containing kanamycin at approximately 50 μg / ml, and cultured overnight at about 30 ° C. After culturing, the cells were diluted approximately 1:10 in standard Termite broth ("TB"); Sambrook et al. , supra) containing kanamycin, and allowed to grow at about 30 ° C at a density of about 0.05-0.8 OD600, at which time the temperature was increased to about 42 ° C to induce the expression and secretion of NT-3opt3. After about 4 hours, about 1 mg of dry cell mass was converted into a pellet and then used boiling for about 10 minutes in about 100 μl of standard SDS-PAGE denaturation / reduction buffer (as described in Example I). Approximately 10 μl of this mixture was then run on a polyacrylamide gel with standard SDS (approximately 18 percent acrylamide). The gel was run at a constant voltage of about 130 volts for about 2.5 hours. The gels, sample buffer and bleed damper were all run from NOVEX, Inc. (San Diego, CA) and used according to the manufacturer's instructions.

B. Analysis of Signal Peptide Nucleic Acid Sequences The efficiency of the different nucleotide sequences encoding the RH signaling peptide was analyzed as the percentage of the amount of processed NT-3opt3 (signaling peptide cleaved from the NT-3opt3 polypeptide) compared to the total amount of NT-3opt3 expressed (NT-3opt3 processed plus unprocessed). To compare the two forms of NT-3opt3, SDS ^ gel was stained with Commassie blue, and then dyed to determine the relative amount of each form. The molecular weight of the unprocessed NT-3opt3 precursor polypeptide is about 15.9 kD, while the molecular weight of the processed NT-3opt3 form is about 13.6 kD. Thus, the two forms of NT-3opt3 showed distinctly different bands on denaturing polyacrylamide gel with SDS. For a number of the RH / NT-3opt3 polypeptides, the bands were visually evident for both Coomassie blue stain gel staining and Western blotting of the gel and detection of the NT-3opt3 gene on Western staining with Polyclonal anti-NT-3opt3 antibody. Two nucleic acid sequences of RH, RH3, RH15, and RHII, appeared by visual inspection to have a high percentage of processed NT-3opt3 compared to the other RH nucleic acid sequences. The sequences of RH3 (SEQ ID NO: 18), RH4 (SEQ ID NO: 19), RH5 (SEQ ID NO: 20), and RH6 (SEQ ID NO: 21) are discussed below; the sequence for the RHll is set forth in Figure 3. RH3: ATGAAGAAACGTGCGCGCGCGATTGCAATTGCAGTAGCGCTGGCTG GCTTTGCTACCGTAGCGCACGCG (SEQ ID NO: 18) RH4: ATGAAAAAGCGCGCACGCGCTATCGCTATCGCTGTTGCTCTGGCTG GCTTTGCAACTGTTGCTCATGCA (SEQ ID NO: 19) RH5:. ATGAAAAAGCGCGCACGCGCAATTGCGATTGCGGTTGCTCTGGCGG GTTTCGCTACCGTTGCGCATGCG (SEQ ID NO-.20) RH6: ATGAAAAAGCGCGCTCGTGCGATCGCGATTGCtGTAGCGCTGGCGG GTTTTGCAACTGTAGCTCACGCG (SEQ ID NO: 21) The gel was screened as described in Example I to determine the relative amounts of processed and unprocessed NT-3opt3 by the different nucleic acid sequences of the RH signaling peptide. The results of the exploration of the activity of the nucleic acid sequences are shown in Table 2 below. The results were reported as the area under the curve (Absorbance x width in millimeters of the protein band) obtained from the gel scan. Table 2 RH3 RH4 RH5 RH6 RHll Unprocessed 0.05 0.87 0.05 0.70 0.05 Processed 0.46 1.01 0.53 0.76 0.40 Percent processed 90.2 53.7 91.4 52.1 88.9 All the literature cited here is specifically incorporated as a reference.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: Amgen Inc. (ii) TITLE OF THE INVENTION: Increased Polypeptide Secretion (iii) SEQUENCE NUMBER: 23 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Amgen Inc., North American Patent Operation / NAO (B) STREET: 1840 Dehavilland Drive (C) CITY: Thousand Oaks (D) STATE: CA (E) COUNTRY: USA ( F) CP: 91320 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM Compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patent in Process # 1.0, Version # 1.25 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: 08 / 215,138 (B) DATE OF SUBMISSION: March 18, 1994 (C) CLASSIFICATION: (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: l Mfct Lys Lys Arg Ala? rg Ala lia Ala lia Ala Val Ala Leu Ala Gly 1 5 10 15 Phß Ala Thr Val Ala His Wing 20 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: ATGA? N? ARC GYGCDCGYOC D? TYGCDATY GCDGTWGCDC TGGCDGGYTT YGCD? CYGTW 60 OCDCAYGCD 69 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE : nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: ATGA ?? AAGC GCGCGCGTGC GATCGCGATC GCGGTTGCGC TGGCTGpCTT CGCTACCGTT 60 GCGCACGCT 69_ (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 363 base pairs, (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: ATGCACTCTG ACCCGGCTCG TCGTGGTGAA CTGTCTGTTT GTGATTCTAT CTCTGAATGG 6C GTTACCGCGG CTG? TA? GAA AACCGCAGTC GACATGTCTG GTGGCACTGT TACCGTCCTC 120 GAGAAAGTTC CTGTATCTAA AGGTCAGCTG AAACAATATT TCTACGAAAC CAAATGCAAT 180 CCGATGGGTT ACACTAAAGA AGGCTGCCGT GGCATCGACA AACGTCATTG 3AACTCTCAG 240 TGTCGTACTA CCCAGTCTTA TGTTCGTGCG CTGACCAT3G ACTCCAAGAA ACGTATCGGC 300 TGGCGCTTCA TCCGTATTGA CACTAGTTGC GTTTGTACTC TGACTATCAA ACGTGGCCGC 360 TAA 363 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 363 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: ATGTACGCTG AACACAAATC CCACCGTGGT GAATATTCCG TTTGCGACTC CGAATCCCTG 60 TGGGTTACCG ACAAATCCTC CGCTATCGAT ATCCGTGGTC ACCAGGTTAC CGTTCTGGGT 120 GAAATCAAAA CCGGTAACTC CCCAGTAAAA CAGTACTTCT ACGAAACCCG TTGCAAAGAA lß? GCTCGTCCGG TTAAAAACGG TTGCCGCGGT ATCGACGACA AACATTGGAA CTCTCAGTGC 24 C AA? ACT? GTC AGACCTACGT TCGTGCTCTG ACCTCCGAAA ACAACAAGCT TGTTGGTTGG 300 CGTTGGATTC GTATCGACAC CAGCTGCGTT TGCGCTCTGT CCCGTAAAAT CGGTCGTACC 360 TAA 363 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ACTGCGGTTT TCTTATCAGC 20 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: CAGCCTTCTT TAGTGTAACC 20 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid, (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: GGATGAAGCG CCAGCCGATA 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple I D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: TTGATTCTAG AAGGAGGA? 19 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear » (ii) TYPE OF MOLECULE: cDNA, (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: TCCGCGGATC CTTAGCGGCC (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: twenty GTTGCTGCGA TTCTCACCAA (2) PAR INFORMATION? SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 103 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: AfTTCTAGA? G GAGGAATAAC ATATGAAAAA GACAGCTATC GCGATTGCAG TGGCACTGGC 60 TGGTTTCGCT ACCGTAGCGC AGGCCCACTC TGACCCGGCT CGT lUj ((2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple ( D) TOPOLOGY: linearF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: AGCGGAGGAT TTGTCGGTA? 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: CTTTGCAACG GGTTTCGTAG_2_° - (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs, (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) MOLECULE TI PO: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: GTTTTCGGAG GTCAGAGCAC 20 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs. (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: TTGATTCTAG AAGGAGGAAT 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear , (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: TCCGCGGATC CTTAGGTACG 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: ATGAAGAAAC GTGCGCGCGC GATTGCAATT GCAGTAGCGC T3GCTGGCTT TGCTACCGTA 60 GCGCACGCG 69 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19: ATGAAAAAGC GCGCACGCGC TATCGCTATC GCTGTTGC'C TGGCTGGCTT TGCAACTGTT 6C GCTCATGCA 69 , (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: ATGAAAAAGC GCGCACGCGC AATTGCGATT GCGGTTGCTC TGGCGGGTTT CGCTACCGTT 60 GCGCATGCG 69 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: ATGAAAAAGC GCGCTCGTGC GATCGCGATT GCTGTAGCGC TGGCGGGTTT TGCAACTGTA GCTCACGCG 69. (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 360 base pairs, (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: CACTCTGACC CGGCTCGTCG TGGTGAACTG TCTGTTTGTG ATTCTATCTC TGAATGGGTT 60 ACCGCGGCTG ATAAGAAAAC CGCAGTCGAC ATGTCTGGTG GCACTGTTAC CGTCCTCGAG 120 AAAGTTCCTG TATCTAAAGG TCAGCTGAAA CAATATTTCT ACGAAACCAA ATGCAATCCG 1TC ATGGGTTACA CTAAAGAAGG CTGCCGTGGC ATCGACAAAC GTCATTGGAA CTCTCAGTGT 240 CGTACTACCC AGTCTTATGT TCGTGCGCTG ACCATGGACT CCAAGAAACG TATCGGCTGG 300 CGCTTCATCC GTATTGACAC TAGTTGCGTT TGTACTCTGA CTATCAAACG TGGCCGCTAA 360 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 360 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: TACGCTGAAC ACAAATCCCA CCGTGGTGAA TATTCCGTTT GCGACTCCGA ATCCCTGTGG 60 GTTACCGACA AATCCTCCGC TATCGATATC CGTGGTCACC AGGTTACCGT TCTGGGTGAA 125 ATCAAAACCG GTAACTCCCC AGTAAAACAG TACTTCTACG AAACCCGTTG CAAAGAAGCT 180 CGTCCGGTTA AAAACGGTTG CCGCGGTATC GACGACAAAC ATTGGAACTC TCAGTGCAAA 240 ACTAGTCAGA CCTACGTTCG TGCTCTGACC TCCGAAAACA ACAAGCTTGT TGGTTGGCGT 30C TGGATTCGTA TCGACACCAG CTGCGTTTGC GCTCTGTCCC GTAAAATCGG TCGTACCTAA 360 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (32)

1. A peptide, characterized in that it comprises the sequence MKKRARAIAIAVALAGFATVAHA (SEQ ID NO: l).

2. The peptide according to claim 1, characterized in that it also comprises in its terminal carboxyl the BDNF polypeptide.

3. The peptide according to claim 1, characterized in that it also comprises in its terminal carboxyl the NT-3 polypeptide.

4. The peptide according to claim 1, characterized in that it also comprises in its terminal carboxyl the NGF polypeptide.

5. The peptide according to claim 1, characterized in that it also comprises in its terminal carboxyl the NT-4 polypeptide.

6. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 2.

• 7. A nucleic acid, characterized in that it comprises a sequence selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; and SEQ ID NO: 21.

8. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 3.

9. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 18.

10. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 19.

11. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 20.

12. A nucleic acid, characterized in that it comprises the sequence of SEQ ID NO: 21.

13. The nucleic acid according to claim 6, characterized in that it also comprises at its 3 'end a nucleic acid encoding a NGF polypeptide without Met.

14. The nucleic acid according to claim 13, characterized in that the NGF polypeptide is BDNF.

15. The nucleic acid according to claim 13, characterized in that the NGF polypeptide is NT-3.

16. The nucleic acid according to claim 13, characterized in that the NGF polypeptide is NGF.

17. The nucleic acid according to claim 13, characterized in that the NGF polypeptide is NT-4.

18. The nucleic acid according to claim 8, characterized in that it also comprises at its 3 'end a nucleic acid encoding a NGF polypeptide without Met.

19. The nucleic acid according to claim 18, characterized in that the NGF polypeptide is BDNF.

20. The nucleic acid according to claim 18, characterized in that the NGF polypeptide is NT-3.

21. The nucleic acid according to claim 18, characterized in that the NGF polypeptide is NGF.

22. The nucleic acid according to claim 18, characterized in that the NGF polypeptide is NT-4.

23. The nucleic acid according to claim 9, characterized in that it also comprises at its 3 'end a nucleic acid encoding the NGF polypeptide without Met.

24. The NGF polypeptide according to claim 23, characterized in that it is NT-3.

25. The nucleic acid according to claim 11, characterized in that it also comprises at its 3 'end a nucleic acid encoding a NGF polypeptide without Met.

26. The NGF polypeptide according to claim 25, characterized in that it is NT-3.

27. A vector, characterized in that it comprises the nucleic acid according to claims 6, 7, 8, 9, 10, 11, or 12.

28. A vector, characterized in that it comprises the nucleic acid according to claims 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

29. A vector characterized in that it is pCFM1656 / BDNFopt3.

30. A vector characterized because it is pCFM1656 / NT-3opt3.

31. A prokaryotic host cell, characterized in that the vector has been inserted in accordance with claims 28, 29 or 30.

32. A method for producing a non-Met form of an NGF polypeptide, characterized in that it comprises: (a) cultivating the prokaryotic host cell according to claims 29, 30, or 31; and (b) separating the secreted NGF polypeptide.