EP0590059A1 - Production et recuperation de neurotrophines recombinees - Google Patents

Production et recuperation de neurotrophines recombinees

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Publication number
EP0590059A1
EP0590059A1 EP92914222A EP92914222A EP0590059A1 EP 0590059 A1 EP0590059 A1 EP 0590059A1 EP 92914222 A EP92914222 A EP 92914222A EP 92914222 A EP92914222 A EP 92914222A EP 0590059 A1 EP0590059 A1 EP 0590059A1
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EP
European Patent Office
Prior art keywords
neurotrophin
denaturing agent
seq
recombinant
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP92914222A
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German (de)
English (en)
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EP0590059A4 (en
Inventor
Nikos Panayotatos
James P. Fandl
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Priority claimed from US07/796,106 external-priority patent/US5389529A/en
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Publication of EP0590059A1 publication Critical patent/EP0590059A1/fr
Publication of EP0590059A4 publication Critical patent/EP0590059A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to the fields of neurobiology, molecular biology and protein biochemistry and, in particular, to neurotrophins and methods of producing and recovering them.
  • the neurotrophins are a group of proteins involved in the functioning of the nervous system. They stimulate the growth of nerve cells and, during embryonic development, support their survival. To date, researchers have identified four neurotrophins: nerve growth factor (NGF) (Ullrich et al., 1983, Nature 30.3:821-825) , brain derived neurotrophic factor (BDNF) (Leibrock et al., 1989, Nature 341:149-152) , neurotrophic factor 3 (NT-3) (Maisonpierre et al., 1990, Science 247:1446-1451) and neurotrophin-4 (Hallbook, et al., 1991, Neuron .6:845-858).
  • NGF nerve growth factor
  • BDNF brain derived neurotrophic factor
  • NT-3 neurotrophic factor 3
  • neurotrophin-4 Hallbook, et al., 1991, Neuron .6:845-858.
  • Nerve growth factor is essential for the development and survival of the peripheral and sympathetic nervous system and the cholinergic neurons of the brain. It is currently being tested for use in Alzheimer's disease. BDNF promotes the survival of sensory neurons in the central nervous system and shows promise as a therapy for Parkinson's disease. NT-3 and NT-4 are newly discovered and their biological role is now being investigated. Like NGF, NT-3 seems to act on sensory and sympathetic neurons.
  • Native human NGF has three subunits: ⁇ , ⁇ and y . Only the ⁇ subunit has neurotrophic activity. /3-NGF is produced
  • SUBSTITUTE SHEET as a pre-pro-protein which is secreted from the cell and processed into the mature form.
  • the pre-pro-peptide contains 187 amino acid residues.
  • the pre- and the pro-sequences are removed, as are two amino acid residues at the C-terminus. This yields a mature protein of 118 amino acid residues.
  • the mature protein has six cysteine residues that form three disulfide bonds.
  • the protein also contains many basic amino acids resulting in a positive charge at neutral pH.
  • the neurotrophins form a family, apparently having evolved from a common ancestral gene.
  • the amino acid sequence of the ⁇ polypeptide of human NGF is 90% homologous to mouse NGF and bovine NGF; and the three human neurotrophins, hNGF, hBDNF and hNT-3, share 60% homology at the amino acid level.
  • the six cysteine residues are conserved in all known neurotrophins, implying that the disulfide bonds are important for function.
  • SUBSTITUTESHEET culture supernatant contained a protein recognized by ⁇ -hNGF antibodies.
  • the partially purified protein exhibited low neurotrophic activity.
  • the level of expression was low. Kanaya et al. stated that these results may have been due either to the failure of the yeast system to remove the two extra C-terminal amino acid residues during maturation or to a problem with folding the hNGF protein.
  • Chan et al. (EP 0 370 171, 1990) produced mature hNGF in insect cells. They fused a gene for pre-pro-hNGF to the polyhedrin promoter and leader sequence of baculoviral DNA. They reported the production of 6 ⁇ g/ml hNGF, but did not physically characterize the product.
  • Mammalian systems provide the most natural environment for the production of mammalian proteins. However, the production of large quantities of proteins in these systems is very expensive. Therefore, there is a need to develop systems that are both less expensive and more productive.
  • One such system is E. coli.
  • Gray and Ullrich (EP 0 121 338, 1984) constructed an expression vector encoding N-methionyl-hNGF and expressed the gene in E. coli. They reported identification of hNGF by
  • Processes for producing recombinant neurotrophins comprise the step of culturing a host cell transformed with a recombinant DNA molecule comprising an expression control sequence operatively linked to a DNA sequence encoding a neurotrophin.
  • the host cells preferably are protease-deficient mutants and, according to the best mode of which e are aware, heat shock regulatory gene mutants.
  • the neurotrophin gene is preferably under the control of a controllable promoter and expression is preferably un-induced or repressed until the cells reach late log phase.
  • the neurotrophins may be also secreted from the cell during production by providing a DNA sequence encoding a signal peptide upstream of the DNA sequence encoding the neurotrophin.
  • Processes for recovering biologically active recombinant neurotrophins produced by host cell cultures comprise the step of solubilizing the neurotrophin in a solution comprising a strong denaturing agent, the solution being essentially free of reducing agents.
  • the solution further comprises a protease inhibitor in an amount effective to inhibit degradation of the neurotrophin by proteases.
  • Other embodiments of the invention further comprise some or all of the following steps: exchanging the strong denaturing agent for a weak denaturing agent; adjusting the solution to comprise a basic amino acid or equivalent at a concentration effective to maintain solubility of the neurotrophin in a non-denaturing environment; purifying the neurotrophin from other molecules in the solution; removing
  • the neurotrophin molecule expressed in E. coli and recovered in a biologically active form is neurotrophin-4.
  • the NT-4 is encoded by a nucleic acid molecule comprising a sequence substantially as set forth for hNT-4 in Figure 11 (SEQ ID NO: 22) or may comprise a sequence that is at least about seventy percent homologous to such a sequence.
  • FIG. 1 is a schematic representation of plasmid pRPN133.
  • the solid line represents pBR322-derived DNA sequences with the origin of replication (ORI) and the / 3-lactamase gene (ampicillin resistance) (Ap) indicated. Distinctive features of the plasmid are indicated by boxed regions with arrowheads indicating the direction of transcription or replication.
  • P L indicates the lambda P L promoter
  • rbsl is the wild type promoter and ribosome binding site of phage T7 l.l.
  • the hNGF gene encodes a mature polypeptide in which the second amino acid residue, serine is replaced by threonine.
  • cI857 indicates the heat inactivatable ⁇ repressor gene.
  • Figure 2 shows dose-response curves to recombinant human NGF produced in E. coli by (A) E8 chick embryo dorsal root ganglia (DRG) explants and (B) dissociated E8 DRG.
  • DRG root ganglia
  • Figure 3 depicts the nucleotide and amino acid sequences of wild type LamB (Fig. 3A, SEQ ID N0:1) and the synthetic LamB signal sequences, LamBl (Fig. 3B, SEQ ID N0:2), LamB2 (Fig. 3C, SEQ ID N0:3), LamB3 (Fig. 3D, SEQ ID N0:4) and LamB4 (Fig. 3E, SEQ ID N0:5).
  • Figure 4 depicts the signal sequence processing kinetics of the modified LamB signal sequences, LamBl and
  • LamB - hBDNF This results in selective transcription of the LamB - hBDNF gene which is placed immediately downstream from the T7 late l.l promoter and ribosome binding site of rbs2.
  • Cells were pulsed with 35 S-methionine for 30 seconds and then chased with an excess, of cold methionine.
  • the cultures were sampled at the indicated times after chase and the labelled proteins were analyzed by SDS-15% PAGE and fluorography. Processing was determined by densitometric scanning of the precursor and mature forms of LamB-hBDNF.
  • FIG. 5 is a schematic representation of pRPN121.
  • the solid line represents pBR322-derived DNA sequences with the origin of replication (ORI) and the ⁇ -lactamase gene (Ap) indicated. Distinctive features of the plasmid are indicated by boxed regions with arrowheads indicating the direction of transcription or replication (ORI) .
  • LacUV5 is the promoter.
  • rbs2 indicates the T7 ⁇ l.l promoter and the T7 $1.1 ribosome binding site with minor nucleotide substitutions from the wild type designed to create convenient restriction sites.
  • LamB2 is the signal sequence.
  • hBDNF yc is the structural gene.
  • Figure 6 depicts a protein profile of Fast S-SEPHAROSE® 1 fractionation of hBDNFmyc.
  • W3110 I q F- /pRPN121 extract after DEAE chromatography was fractionated as described on a 1.6 cm X 6.5 cm Fast S-SEPHAROSE® column in 7 M urea, 50 mM histidine, pH 5.0, 1 mM EDTA. Fractions as indicated were analyzed by SDS-15% PAGE and proteins
  • Figure 7 is a summary of the purification procedure.
  • Strain W3110 I q F"/pRPN121 was grown, induced, and extracted as described above. Pooled column fractions were analyzed by SDS-PAGE on a 15% acrylamide gel and stained with Coomassie Blue. Lane 1, DEAE; lane 2, Fast S-SEPHAROSE® 1; lane 3, Fast S-SEPHAROSE® 2; lane 4, C4 reverse phase HPLC. Molecular weight standards are indicated.
  • Figure 8 depicts a dose response curve for purified hBDNFmyc stimulation of E8 chicken embryo DRG neurite outgrowth.
  • the hBDNFmyc activity is compared with recombinant hBDNF purified from a Chinese hamster ovary cell line. Both proteins were purified to greater than 95% by C4 reverse phase HPLC.
  • Figure 9 depicts a dose response curve for the stimulation of E8 chicken embryo DRG neurite outgrowth by recombinant hBDNF purified from E. coli.
  • the hBDNF was purified to greater than 95% by C4 reverse phase HPLC.
  • FIG 10 is a schematic representation of pRPN149.
  • the solid line represents pBR322-derived DNA sequences with the origin of replication (ORI) and the /3-lactamase gene (Ap) indicated. Distinctive features of the plasmid are indicated by boxed regions with arrowheads indicating the direction of transcription or replication (ORI) .
  • LacUV5 is the promoter.
  • rbs2 indicates the T7 ⁇ l.l promoter and the T7 ⁇ l.l ribosome binding site with minor nucleotide substitutions from the wild type designed to create convenient restriction sites.
  • LamBl is the signal sequence.
  • hBDNF is the structural gene.
  • Figure 11 is the DNA sequence of a portion of the isolated human genomic phage clone 7-2 encoding human NT-4 (SEQ ID NO:22; ATCC Accession No:75070) .
  • the predicted hNT-4 protein encoded by the genomic clone 7-2 is represented by the one-letter symbols for amino acids (SEQ ID NO:22; ATCC Accession No:75070) .
  • the boxed region represents the predicted cleavage site of the hNT-4 preprotein. Arrows indicate conserved residues in the presequence.
  • the underlined region (N-R-S) represents a consensus sequence for N-glycosylation.
  • the circled region represents the initiating methionine.
  • the splice acceptor site is located at base pair 461-462 (AG) , representing the 3 '-end of the intron.
  • Figure 12 is a schematic representation of pRG91.
  • the solid line represents pBR322-derived DNA sequences with the origin of replication (ORI) and the 3-lactamase gene indicated. Distinctive features of the vector are shown by boxed regions.
  • LacUV5 is the promoter.
  • rbs2 is the phage T7 ⁇ l.l promoter and the T7 ⁇ l.l ribosome binding site.
  • LamB2 is the signal sequence.
  • Plasmid pRG91 (Regeneron Pharmaceuticals) is a pBR322- based vector designated for the expression of recombinant proteins and their secretion into the periplasmic space of Escherichia coli.
  • the vector consists of the strong, regulated, lacUV5 promoter followed by the phage T7 ⁇ l.l promoter and ribosome binding site inserted between the EcoRI and Nrul restriction sites in pBR322. These control elements direct the expression of the LamB2 signal sequence to which recombinant protein gene sequences may be fused.
  • the DNA sequences between the unique Nrul and PvuII restriction sites were deleted, resulting in increased plasmid copy number. This plasmid confers ampicillin (Ap) resistance:
  • Figure 13 is a dose response curve for hNT-4 expressed in E. coli. Soluble protein extract from an induced culture of strain RFJ26 containing plasmid pRG173 was assayed on E8 dorsal root ganglion explants. The background activity of this assay was 0.2 units.
  • Figure 14 depicts the effect of hNT-4 on CAT activity. Treatment of motor neuron enriched cultures with a
  • SUBSTITUTE SHEET partially purified extract from an induced culture of strain RFJ26 containing plasmid pRG173 resulted in a 3.6- fold (at 1:20 dilution) increase in CAT activity after 48 hours as compared to untreated (C-NT) and buffer (C-buffer) controls.
  • the E. coli extract was passed through a Sepharose-S column as disclosed infra prior to treatment of motor neuron enriched cultures.
  • neurotrophin refers to any naturally occurring member of the neurotrophin family. This includes naturally occurring proteins sharing amino acid sequence homology with any known neurotrophin and conserving the six characteristic cysteine residues. (Some of these proteins may fall structurally into the neurotrophin family, yet may exhibit biological activity other than neurotrophic activity.)
  • neurotrophin also refers to engineered neurotrophins whose amino acid sequences are derived from or patterned after the naturally occurring neurotrophins.
  • neurotrophins comprising amino acid sequences from different neurotrophins (e.g., NGF and NT-3) or from the same neurotrophin of different species (e.g., hBNDF and pig BDNF) ; neurotrophins whose genetic sequences have point substitutions, addition or deletion mutations; neurotrophins derived from the pre-pro-sequence (e.g., neurotrophins having the two carboxy-terminal amino acid residues encoded by the codons immediately preceding the stop codon of the native gene ("full length neurotrophin”)); and includes fragments of a neurotrophin which exhibit neurotrophic activity (e.g., those whose first six amino acids are altered or deleted) .
  • neurotrophins e.g., NGF and NT-3
  • hBNDF and pig BDNF neurotrophins whose genetic sequences have point substitutions, addition or deletion mutations
  • neurotrophins derived from the pre-pro-sequence e.g., neurotroph
  • This invention provides processes for producing and recovering biologically active recombinant neurotrophins.
  • a recombinant protein as used in this specification, is a protein expressed from a recombinant DNA molecule in a host cell transformed with it.
  • a recombinant DNA molecule is a hybrid molecule comprising DNA sequences from different sources that have been joined together. Sambrook et al. (1989, Molecular Cloning: A Laboratory Manuel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor) describes many conventional techniques for recombinant DNA technology.
  • producing recombinant neurotrophins involves culturing a non-animal host cell transformed with a recombinant DNA molecule having an expression control sequence operatively linked to a DNA sequence encoding a neurotrophin.
  • An expression control sequence is operatively linked to a DNA sequence encoding a polypeptide when the expression control sequence directs and promotes the transcription and translation of the DNA sequence.
  • Culturing a transformed host cell involves incubating the cell in culture conditions appropriate for the growth of the cell and the expression of the DNA sequence.
  • DNA sequences encoding neurotrophins are available from several sources.
  • the literature discloses DNA sequences for the four known human neurotrophins (i.e., hNGF (Gray et al., EP 0 121 338, 1984), hBDNF (Hyman et al., WO 91/03568), hNT-3 (Hohn et al., WO 91/03569) and Xenopus NT-4 (Hallbook, et al., 1991, Neuron 6:845-858) and for many non-human neurotrophins.
  • hNGF Gram et al., EP 0 121 338, 1984
  • hBDNF Hemaset al., WO 91/03568
  • hNT-3 Hohn et al., WO 91/03569
  • Xenopus NT-4 Hallbook, et al., 1991, Neuron 6:845-858
  • Genomic libraries are suitable sources of DNA
  • SUBSTITUTESHEET may also screen cDNA and genomic libraries using any of the methods known to the art to identify and isolate DNA sequences encoding neurotrophins. Alternatively, one may construct synthetic or semi-synthetic genes using conventional DNA synthesizers. Undoubtedly, researchers will discover DNA sequences encoding new neurotrophins. The methods of this invention will be useful for producing and recovering these neurotrophins, as well.
  • the invention relates generally to production of NT-4 or a derivative or fragment thereof by growing a recombinant bacterium containing a nucleic acid encoding NT-4 or derivative or fragment under conditions such that the NT-4 or derivative or fragment thereof is expressed by the bacterium, and recovering the produced NT- 4 or NT-4 derivative or fragment.
  • the NT-4 is human NT-4.
  • an NT-4 derivative which is a chimeric or fusion protein is produced.
  • the produced NT-4, or NT-4 derivative or fragment is biologically active, i.e., capable of exhibiting one or more of the known functional activities of NT-4, as assayed by any methods known in the art or taught herein (e.g., in vitro assays of the ability to promote outgrowth in E8 DRG explants, the ability to stimulate CAT activity in purified motor neuron cultures; see Section 9 , infra) .
  • sequences encoding human NT-4 are expressed in an E. coli expression system and a purification scheme as disclosed infra is used to produce useful amounts of human NT-4.
  • the nucleic acid encoding human NT-4 which is thus expressed can be that contained in nucleic acid pRG173 (ATCC Accession Number 75131) or HG7-2 (ATCC Accession Number 75070) or shown in Figure 11 (SEQ ID NO:22), or isolated by any methods known in the art, or as follows: Mixtures of 5' and 3' oligonucleotides representing all possible codons
  • SUBSTITUTE SHEET corresponding to known NT-4 sequences or to conserved amino acid sequences from known neurotrophins are utilized as primers in the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • Primary and secondary PCR amplification reactions of human (or other mammalian) cDNA or genomic libraries result in the isolation of a PCR product that can be utilized as 32 P-labelled probes to isolate a full length cDNA or genomic clone encoding NT-4.
  • human neurotrophin-4 should be understood as meaning any human homologue of the Xenopus NT-4 (Hallbook, et al., 1991, Neuron 6:845-858) , including a distinct yet homologous (e.g., at least about seventy percent homology) neurotrophin molecule.
  • the literature discloses a variety of expression control sequences useful for expressing DNA sequences in transformed non-animal hosts. These include, among others, in bacteria, the lac system, the trp system, the TAC system, the TRC system, the lambda P L promoter, the T7 late promoters, and the control regions of the fd coat protein; and in yeast, the phosphoglycerate kinase promoter, the Gal 4 promoter, the His promoter, the alcohol dehydrogenase promoter, the alkaline phosphatase promoter and the ct- ating factor promoter. Controllable expression control sequences are preferable and, among these, a temperature inducible lambda P L promoter, the lacUV5 promoter and the T7 ⁇ l.l promoter are most preferable for expression in E. coli.
  • the literature also discloses a variety of expression vector/host systems suitable for bacterial and fungal hosts, including plasmids, bacteriophages, cosmids and derivatives of them. Examples of these systems are, for bacteria, col Fl, pCRl, pBR322, pMB9, RP4, phage lambda, M13 and filamentous viruses; and, for yeast, 2 ⁇ .
  • Preferred plasmids in bacteria are stable in the host cell and present at medium copy numbers of 20-200 copies per cell.
  • SUBSTITUTE SHEET We used plasmids derived from pBR322, such as those described by Panayotatos (1987, Engineering an Efficient Expression System, in: Plasmids-A Practical Approach, ed. Herdy, K. , IRL Press, Oxford/Washington, D.C.). In particular, we prefer plasmids of the RPN class, developed by Regeneron Pharmaceuticals, Inc. and described more fully below.
  • E. coli Bacillus, Strepto vces, Saccharomyces and Pichia pastoris. We prefer E. coli.
  • Culturing transformed host cells results in the expression of neurotrophins.
  • neurotrophins expressed in bacteria are subject to degradation by intracellular proteases, especially those induced as part of the "heat shock" response to foreign proteins (Goff et al., 1984, Proc. Natl. Acad. Sci. USA .81:6647-6651). Therefore, we prefer to use protease-deficient mutants as host cells.
  • the expression of the heat shock genes is regulated by heat shock regulatory genes, such as the htpR gene of E. coli. Mutants of the HtpR gene are deficient in expression of heat shock protease genes, as well as other genes that contribute to the heat shock response.
  • HtpR' Ion' double mutants are particularly useful.
  • U.S. patent 4,758,512 Goldberg et al. describes other suitable strains.
  • Another preferred E. coli strain is RFJ26.
  • Neurotrophins are toxic to bacterial cells that express them. Therefore, in order to maximize yield, we prefer to induce neurotrophin expression only in dense
  • SUBSTITUTE SHEET bacterial cultures for example, cultures in late log phase.
  • We typically induce production of neurotrophins by shifting growth temperature to 42°C for 30 minutes and continuing incubation for 3-20 hours at 38°-42°C. Inducing expression for more than 16 hours in actively growing cells eventually causes cell death.
  • the inability of E. coli to accumulate neurotrophins may be the result of one or more properties of the neurotrophin gene or protein.
  • the structure of the neurotrophin mRNA, particularly the structure proximal to the translation start point, may prevent efficient translation.
  • the neurotrophin may prevent its own synthesis by interacting directly with its mRNA, or it may interact directly or indirectly with some component of the DNA replication/transcription/translation machinery of E. coli.
  • a signal sequence gene fused to the 5* end of the neurotrophin gene may provide a nucleotide sequence proximal to the translation startpoint that is more conducive to efficient translation, thus resulting in higher levels of neurotrophin accumulation.
  • sequestering the neurotrophin in the periplasmic space prevents it from interfering with any cytosolic component necessary for protein synthesis. It also protects it from attack by cytosolic proteases. It is also possible that the secretion of a mature neurotrophin into the periplasmic space may provide an environment more conducive to the proper folding of the protein.
  • a signal sequence is provided by constructing a recombinant DNA molecule in which the DNA sequence encoding the neurotrophin comprises, from 5* to 3' , a fused gene encoding a signal or leader sequence appropriate to the host cell which is in-frame with a DNA sequence for the neurotrophin.
  • the literature describes several signal sequences useful in such constructions. For example, LamB, O pA and PhoA are useful in E. coli. (Denefle et al., 1985, Gene .85:499-510; Wong et al., 1988, Gene 658.:193-203) . We prefer LamB and, in particular, modified LamB signal sequences that improve the translational efficiency of the LamB mRNA.
  • LamB has a hydrophobic core of 10 amino acid residues. Mutational analysis of several E. coli signal sequences suggests that the length of the hydrophobic core region can have a strong effect on signal sequence activity. We have found that increasing the length of the hydrophobic region by the addition of up to ten hydrophobic amino acid residues improves the efficiency of processing LamB fusion precursor polypeptides. Fewer than six is preferable and four is most preferable. The choice of hydrophobic amino acids added is not critical, nor is the precise location at which they are added to the
  • the recombinant neurotrophins are released from the culture of host cells by harvesting the cells, lysing them, centrifuging the lysate and collecting the lysate pellet.
  • the art is familiar with many techniques to lyse cells including enzymatic (e.g., lysozy e) , chemical (e.g., alkali, SDS) and mechanical (e.g., French press, hydrodynamic shear) .
  • This invention further provides processes for recovering the recombinant neurotrophins produced in cell cultures. These processes overcome the problems associated with conventional methods of recovering recombinant proteins that have been applied to neurotrophins.
  • Native neurotrophins are soluble in neutral buffers. However, recombinant neurotrophins from E. coli behave as insoluble proteins. Recombinant neurotrophins have been detected in the cytosolic fraction and, when exported, have been recovered from the periplasmic space using standard techniques such as osmotic shock, spheroplasting, or freeze-thaw (Bochner et al., U.S patent 4,680,262).
  • the literature describes standard techniques for purifying recombinant proteins from inclusion bodies (see, e.g., Builder et al. , U.S. patent 4,620,948; Hershenson et al., U.S. patent 4,961,969; and Hung et al. , U.S patent 4,734,362). These techniques involve dissolving the protein in a solution comprising a strong denaturant and a reducing agent. After exchanging the strong denaturant for a weak denaturant, the proteins are renatured in a neutral solution and oxidized to form the correct disulfide bonds. The majority of the recombinant neurotrophin produced by these methods is biologically inactive.
  • the process involves solubilizing the neurotrophin in a strong denaturing agent while maintaining the correct oxidation state of the protein by avoiding the use of reducing agents. Reducing the disulfide bonds during purification destroys the
  • our process for recovering recombinant neurotrophins produced by a host cell culture comprises the step of denaturing the neurotrophin by dissolving it in a solution comprising a strong denaturing agent which solution is also essentially free of reducing agents.
  • Denaturing agents as used herein refer to compounds which, in aqueous solution, reversibly unfold dissolved proteins by at least partially eliminating tertiary and secondary structure through the disruption of hydrogen bonds or alteration of the thermodynamic surroundings of the protein.
  • Strong denaturing solutions include guanidinium salts (e.g., guanidinium hydrochloride) and alkali metal thiocyanates (e.g., sodium thiocyanate) at
  • the preferred denaturing solutions are 7 M - 9 M guanicinium HCl.
  • Preferred conditions are pH 7.0 - pH 9.0 and room temperature.
  • Most preferred is 8 M guanidinium HCl, pH 8.0.
  • the solution must be essentially free of reducing agents.
  • a solution essentially free of reducing agents is one in which a protein's disulfide bonds are maintained. Addition of even small amounts of disulfide reducing agents, such as jS-mercaptoethanol or dithiothreitol, during the practice of this invention will destroy the activity of the purified neurotrophin.
  • Recombinant neurotrophins apparently are subject to degradation by metalloproteinases. Therefore, preferably, one recovers neurotrophins in solutions comprising metalloproteinase inhibitors.
  • heavy metal chelators such as EDTA.
  • concentration of EDTA may range from a minimum of at least 1 mM to a maximum of about 200 mM. Concentrations of 5 mM - 80 mM are preferable and 50 mM is most preferable. After chelated heavy metal ions have been dialyzed from the solution, EDTA may be reduced or eliminated. Langley et al., 1990, EP 0 398 753, describes peptides useful as metalloproteinase inhibitors.
  • the recovery process may also comprise one or more of the following steps.
  • weak denaturing solutions include urea at 4 M - 9 M and, preferably, 6 M - 8 M, pH 7.0 - pH 9.0, at room temperature.
  • the most preferable weak denaturing solution is 7 M urea, 50 mM Tris-HCl, 10 mM NaCl, 5 mM EDTA, pH 8.0. Dialysis is the preferred method
  • the weak denaturing agent of this step is preferably urea of lower concentration.
  • Another step in the recovery process is purifying the recombinant neurotrophin from other contaminants in the solution.
  • Any of the typical protein isolation techniques known to the art may be used.
  • the isolation step can begin at any stage after solubilization of the neurotrophin and may continue through the denaturing agent exchange and removal steps.
  • we begin purification at the urea phase because this agent does not interfere with ion exchange chromatography and because partial purification made the neurotrophin easier to dissolve in the non-denaturing solution.
  • Solubility depends upon degree of purity of the neurotrophin as well as the character of the other contaminants in solution. For example, negatively charged molecules such as DNA will interfere with solubility of even very pure neurotrophin. Therefore, we maintain the solubility of the neurotrophin in the non-denaturing solution by adjusting the solution to comprise a basic amino acid or an equivalent, such as indole acetic acid. The concentration of this species should be effective to maintain the neurotrophin in solution. Solutions of basic amino acids include
  • results indicate that our method produces uniform neurotrophin molecules, consistently having the same N-terminal amino acid.
  • expression of neurotrophins in the mammalian CHO cell system produces a mixture of neurotrophin molecules with varying N-terminal amino acids. Therefore, the recombinant neurotrophins produced by our process appear to be unique.
  • the processes of this invention are also useful for recovering other proteins having biochemical properties similar to the neurotrophins. That is, the processes are useful for recovering proteins that fold correctly in bacteria and form the proper disulfide bonds, but, once denatured and reduced in conventional recovery techniques, are very difficult to renature with proper disulfide bonds. This includes proteins with pH greater than about 9.0 and having at least two disulfide bonds.
  • Candidate molecules include secretory leukocyte protease inhibitor (Miller et al., 1989, J. Bacteriology 171:2166-2172) and full length recombinant CD4 (Fisher et al. , 1989 WO 89101940) .)
  • the present invention discloses the expression of biologically active human neurotrophin-4.
  • the human NT-4 DNA sequence was subcloned into the DNA plasmid vector pRG91, resulting in pRG173.
  • This hNT-4 containing plasmid was transformed into E. coli strain RFJ26, and methods described in the instant specification were utilized to recover biologically active NT-4 from the culture system.
  • applicants are not to be limited to such a specific embodiment. For example,
  • SUBSTITUTESHEET any nucleic acid sequence substantially homologous to the region of HG7-2 encoding human NT-4 can be utilized to construct any number of DNA plasmid expression vectors as described throughout the specification or known to the skilled artisan, which in turn can be utilized to transform any number of E. coli bacterial strains in order to produce useful amounts of biologically active NT-4.
  • a DNA sequence carrying the mature human NGF (hNGF) gene was amplified from human genomic DNA using known human
  • NGF sequences (Nancy Ip; Regeneron Pharmaceuticals, Inc.) by PCR amplification.
  • PAN-20 5'-AAGCGGTCGA CATCTOATCC AATCTTCCAT
  • pRPN50 is derived from expression plasmid pNKS97 (Panayotatos, 1987, Engineering an Efficient Expression System, In: Plasmids-A Practical Approach, ed. Herdy, K. , IRL Press, Oxford/Washington, D.C.) by the insertion of the lambda P L promoter" at the promotor insertion site.
  • the cI857 lambda P L repressor gene was incorporated into pRPN102 to facilitate repression at 30°C and to allow derepression by heat shock at 42°C.
  • This DNA sequence was PCR amplified using the primers EVD-26 (5•-CCATTATCGC . GACCAGAGGT-3•) (SEQ ID NO:9) and EVD-27 (5'-TCTTGCTCGC GAGTTATCAG CTATGCG-3') (SEQ ID NO:10) that generate a Nrul restriction site at each end.
  • This 821 bp fragment extends 70 bp upstream from the cI857 coding sequence in a region that contains the PRM constitutive promoter as well as 38 bp beyond the cI857 termination codon.
  • Plasmid pRPN133 has been deposited with the ATCC and has been assigned Accession No: 75029.
  • Cells were harvested by centrifugation and the cell pellets stored at -20°C.
  • Cell pellets (1.0 - 2.5 g) were thawed, resuspended in 20.0 mL buffer A (100 mM Tris-HCl, 50 mM EDTA, pH 8.0), passed through a STANSTED® cell disrupter at 10,000 psi, and centrifuged at 23,000 x g for 15 minutes at 4°C. The pellet was washed twice with 10.0 mL of 2 M guanidinium-HCl, 5 mM EDTA, pH 8.0.
  • the supernatant was dialyzed against 2 liters buffer B (7 M urea, 100 mM histidine, 0.1 mM EDTA, pH 6.0) with two buffer changes at 25°C for 20 hours.
  • the dialysate was centrifuged at 23,000 x g for 15 minutes at 4°C.
  • the pooled fractions were dialysed against 100-fold excess volume buffer C (7 M urea, 50 mM Tris-HCl, 0.1 mM EDTA, pH 8.5) at 25°C for 20 hours.
  • the dialysate was applied to a DEAE-SEPHAROSE column (2.5 x 7.5 cm d x h) equilibrated in buffer C at a flow rate of 2.0 mL per minute.
  • the flow-through fractions containing the hNGF were pooled and dialyzed twice against 40 volumes of 100 mM histidine, 0.1 mM EDTA, pH 6.0 at 4°C overnight.
  • the dialysate was centrifuged at 23,000 x g for 15 minutes at 4°C.
  • the dialysate was then applied to a TOYOPEARL CM 6505® (weak cation exchange) column (1.0 x 5.0 cm) equilibrated with 100 mM histidine, 0.1 mM EDTA, pH 6.0
  • the biological activity of the purified protein was tested for neurite outgrowth in E8 explanted and dissociated dorsal root ganglia (Fig. 2A and 2B) (Lindsay et al. , 1985, Dev. Biol. 112:319-328) .
  • the recombinant hNGF purified from E. coli by this method was found to be as active as NGF purified from mouse salivary gland.
  • SEQ ID N0:1 is a naturally occurring E. coli signal sequence which was selected for the construction of a series of secretion vectors based on the pRPN series of expression vectors developed at Regeneron Pharmaceuticals,
  • LamB was inserted into the structural gene insert site such that expression of LamB is under control of the lacUV5 or
  • LamBl SUBSTITUTESHEET LamB signal sequence. However, we altered the nucleotide sequence relative to the wild type nucleotide sequence for the purpose of constructing unique restriction enzyme sites and for the maximization of translation efficiency. LamBl also has seven degenerate nucleotide substitutions replacing C or G with A or T. This reduces the stability of possible secondary structure of mRNA. LamBl also has several new restriction sites.
  • LamB2 (Fig. 3C, SEQ ID NO:3) as a modification of LamBl.
  • To make LamB2 we made nucleotide changes to the 3' end of LamBl by PCR amplification. These changes introduced an Ea ⁇ l restriction site which facilitates the cloning of blunt end DNA fragments.
  • the fusion of mature hBDNF to LamBl results in efficient synthesis of the fusion protein in E. coli.
  • the authenticity of the synthesized product was confirmed by selective synthesis in a DNA-dependent coupled transcription-translation cell-free protein synthesis system, by the selective synthesis of the product using a T7 RNA polymerase expression system in E. coli. and by the synthesis of the product with a C-terminal myc tag allowing for identification of the chimera with a myc-specific monoclonal antibody.
  • Either one of these fusion proteins synthesized in E. coli was processed to mature hBDNF as evidenced by its mobility on SDS-PAGE.
  • the level of expression obtained with LamBl or LamB2 results in accumulation of hBDNF from about 1% to 10% of total cell protein.
  • LamBl was modified to increase the efficiency of processing.
  • Four amino acids (Leu-Ala-Val-Leu, or LAVL) were inserted into the Nhel site of LamBl to yield LamB3 (Fig. 3D, SEQ ID N0:4).
  • the same insertion was made in LamB2 to yield LamB4 (Fig. 3E, SEQ ID N0:5). This insertion results in extension of the hydrophobic core region of LamBl and LamB2 from 10 to 14 amino acids.
  • SUBSTITUTESHEET results in a 5-fold increase in the rate of processing of the hBDNF fusion protein into mature hBDNF (Fig. 4) .
  • LamB3- and LamB4-hBDNF molecules that are exported into the periplasm are processed into mature hBDNF more rapidly than wild type LamB fusions. However, fewer molecules are exported, so the net amount of mature hBDNF in this case is not increased. In any case the increased translocation efficiency of LamB3 and LamB4 should result in improved yields of other proteins.
  • LamBl signal sequence fragment was constructed as two complementary synthetic oligonucleotides (LamB80, 5'-ATGATCACAC TGCGTAAGCT TCCGCCTAGCT GTAGCAGTAG CAGCAGGTGT AATGTCTGCA CAGGCCATGG CCCGGGATCC-3 • (SEQ ID NO:11) and LamB88, 5 « -CTAGGGATCC CGGGCCATGG CCTGTGCAGA CATTACACCT GCTGCTACTG CTACAGCTAG CGGAAGCTTA CGCAGTGTGA TCATCATG-3' (SEQ ID N0:12)), designed so as to generate upon annealing protruding ends corresponding to those of the Spel and SphI restriction sites.
  • This DNA fragment was ligated into the SphI and Spel restriction sites of pRPN16 (Regeneron Pharmaceuticals, Inc.) resulting in plasmid pRPN52.
  • This plasmid was subsequently modified, for the purpose of creating additional restriction sites, by the insertion of a synthetic DNA fragment, consisting of the annealed product of the complementary LamB2A, (5'-CATGGCCAGT CGGCCGAG-3' (SEQ ID NO:13)) and LamB2B (5 « -GATCCTCGGC CGACTGGC-3• (SEQ ID NO:14)) oligonucleotides, between the unique Ncol and BamHI restriction sites in the LamBl signal sequence in pRPN52.
  • the resulting plasmid, pRPN88 contains the LamB2 signal sequence with unique restriction sites at the signal peptidase recognition sequence to facilitate fusion of the signal sequence to any other DNA sequence.
  • the LamB2 signal sequence in pRPN88 is under transcriptional control of the lacUV5 or the T7 ⁇ l.l promoter and translational control of the T7 ⁇ l.l ribosome binding site.
  • Human BDNFmyc is a protein comprising, from N- to C-terminus, mature hBDNF fused to an antigenic "tag.”
  • the tag is a peptide having the amino acid sequence EQKLISEEDL (SEQ ID NO:15) These ten amino acid residues derive from the human c-myc proto-oncogene.
  • Antibodies recognizing this tag are useful for identifying hBDNFmyc in a sample (Evan et al., Mol. Cell. Biol. 5_:3610-3616; see also, S. Squinto et al. , "Assay Systems for Detecting Neurotrophic Activity," U.S. application 07/5321,283, incorporated herein by reference) .
  • the hBDNFmyc DNA sequence was PCR amplified from pCDM8-hBDNFmyc (Regeneron Pharmaceuticals, Inc.) using oligonucleotide primers N8-hBDNF (5'-CCCACTCTGA CCCTGCCCGC CGAGGG-3 ' (SEQ ID NO:16)) and C2-hBDNFmyc (5'-GCTATGCGGC CGCTACAGAT CCTCCTC-3 1 (SEQ ID NO:17).
  • the amplified DNA fragment was digested with Ea ⁇ l and cloned into the Ball and EagI sites of the LamB2 sequence in pRPN88, resulting in plasmid pRPN98.
  • DNA sequences encoding mature hBDNF are isolated by PCR amplification as described. (Hyman et al., 1991, WO 091/01568.)
  • a double stranded DNA sequence encoding the ten-amino acid myc peptide tag may be chemically synthesized.
  • the hybrid hBDNFmyc DNA sequence is then provided with Ball and EagI restriction sites at the 5' and 3' ends, respectively.
  • LamB2 signal sequence is fused to the mature part of the hBDNFmyc protein so that cleavage at the signal peptidase recognition sequence should yield hBDNFmyc protein starting at the histidine residue at +1 relative to the pro-protein processing site (Leibrock, 1989, Nature 341:149-152).
  • Plasmid pRPN 121 has been deposited with the ATCC and been assigned Accession No: 75028.
  • the cell pellet (approximately 5 g) was thawed and resuspended in 20 mL 0.2 M Tris-HCl, pH 8.0, 1 mM CaCl,, and 25 units micrococcal nuclease (Boehringer Mannheim) .
  • the cell suspension was passed through a French pressure cell at 8000 psi and then centrifuged at 15,000 rpm for 15 minutes in a SA600 rotor at 4°C.
  • the pellet was resuspended in 30 mL of 0.2 M Tris-HCl, pH 8.0, 10 mM EDTA, 2% Triton X-100 and gently rocked at room temperature for one hour then centrifuged at 15,000 rpm for 15 minutes in a SA600® rotor at 4°C. The pellet was washed twice in 20 mL of 2 M guanidinium-HCl. The pellet was resuspended in 10 mL of 8 M guanidinium-HCl, 10 mM Tris-HCl, pH 8.5, 10 mM
  • the extract was dialyzed overnight at room temperature against 100 X volume 7 M urea, 50 mM Tris-HCl, pH 8.5, 10 mM NaCl, 1 mM EDTA.
  • the dialysate was applied to a DEAE ZETA-PREP® disk (Cuno, Inc., Meriden, CT) equilibrated in 7 M urea, 50 mM Tris-HCl, pH 8.5, l mM EDTA, at a flow rate of 3 mL/minute and washed to baseline with the same buffer.
  • the flow-through fractions were brought to 50 mM histidine, pH 5.0 then dialyzed overnight at 4°C against 100 x volume 7 M urea, 50 mM histidine, pH 5.0, l mM EDTA.
  • the dialysate was applied to a 1.6 cm x 6.5 cm column of S-SEPHAROSE® equilibrated with 7 M urea, 50 mM histidine, pH 5.0, 1 mM EDTA, at a flow rate of 1 mL/minute, and washed to baseline with the same buffer.
  • Proteins were eluted with a NaCl gradient from 0.0 M - 1.0 M in 200 ml. Fractions containing hBDNFmyc were collected (Fig. 6) .
  • Fractions containing hBDNFmyc were dialyzed against 200 X volume 50 mM histidine, pH 5.0, 50 mM NaCl, l mM EDTA. The dialysate was applied to a 1.6 cm X 1.5 cm column of S-SEPHAROSE® equilibrated with 50 mM histidine, pH 5.0, 50 mM NaCl, 1 mM EDTA, at a flow rate of 1 mL/minute and washed to baseline with the same buffer. Proteins were eluted with a NaCl gradient from 0.0 M - 1.0 M in 200 ml. The fractions containing hBDNFmyc were collected and pooled.
  • the hBDNYmyc in this sample (approximately 85% pure) was applied directly to a 0.45 cm X 5.0 cm C4 reverse phase column (VYDAC®) and eluted with a gradient of 0-67% acetonitrile in 0.1% trifluoroacetic acid in 80 mL at a flow rate of 0.75 mL/minute.
  • the peak fraction was greater than 95% pure (Fig. 7) .
  • the purified hBDNFmyc was biologically active in promoting neurite outgrowth from dorsal root ganglion (DRG) explants and nodose ganglion explants from E8 chicken embryos, as well promoting survival and dendritic outgrowth of dissociated neurons from E8 chicken DRG's (Fig. 8) .
  • This activity is comparable to recombinant human BDNF purified from mammalian cell extracts on DRG explant assays. Material assayed corresponds to Fig. 7, lane 4.
  • hBDNFmyc The process described for the production and recovery of hBDNFmyc was also used to produce and recover recombinant mature full length hBDNF, and yielded protein with similar bioactivity (Fig. 9) .
  • the construction of plasmid pRPN149 (Fig. 10) which expresses a LamBl-hBDNF fusion protein, is analogous to the construction of pRPN121.
  • the synthetic LamBl DNA fragment, described above, was cloned into the SphI and Spel restriction sites of pRPN09 (Regeneron Pharmaceuticals, Inc.) resulting in plasmid pRPN31.
  • the mature hBDNF DNA sequence was PCR amplified from pCDM8-hBDNF (Regeneron Pharmaceuticals,
  • Hindlll-BamHI fragment including the LamBl-hBDNF fusion gene was subsequently cloned into the Hindlll-BamHI restriction sites of pRPN52 (described above) resulting in plasmid pRPN66.
  • the NruI-PvuII deletion of pBR322 sequences resulting in higher copy number was made in pRPN66 resulting in plasmid pRPN149.
  • expression of LamBl-hBDNF is under control of
  • Plasmid pRPN149 has been deposited with the ATCC and has been assigned Accession No: 75027.
  • Human NT-3 is produced and recovered by processes similar to those we described for hNGF and hBDNF.
  • a DNA sequence encoding mature full length hNT-3 is PCR amplified from a cDNA library from human brain cells (Hohn et al., 1991, WO 91/03569, incorporated herein by reference).
  • the oligonucleotide primers EVD-45 (5'- CCTATGCAGA GCATAAGAGT CACCGAGGA-3 » ) (SEQ ID NO:20) and EVD-7 ( ⁇ '-GTAAGGGCGG CCGAAGTTTA ATAAATAAAG GTC-3 » ) (SEQ ID NO:21) are used. These primers are derived from the DNA sequence for rat NT-3 (Maisonpierre et al. , Science 247:1446-1451) .
  • the sense primer is nearly identical to the human NT-3 sequence.
  • the antisense primer hybridizes approximately one hundred base pairs downstream of the termination codon of the human gene.
  • This DNA fragment has a C-terminal EagI restriction site suitable for insertion into Ball and EagI sites of pRPN88, where it would replace the hBDNF DNA sequence.
  • the resulting plasmid is used to transform E. coli I q F-W3110.
  • recombinant hNT-3 is produced and recovered as in Example 7.
  • We expect recombinant hNT-3 purified from E. coli to exhibit neurotrophin activity similar to that described by Hohn et al., 1991, (WO 91/03569).
  • the DNA sequence encoding the putative mature region of the human NT-4 (hNT-4) gene was amplified by PCR from NT-4 HG7-2 DNA, using the N1-NT4
  • C1-NT4 (5'-CCGGGGTCTCTGAAACTGCACCAGCGAGTCG-3 ') [SEQ ID NO:23] and C1-NT4 (5 « -GGTGCAGTTTCAGAGACCCCCATACGCCGGCTGCGGTTGGC-3 ') [SEQ ID NO:24] oligonucleotides as primers.
  • the C1-NT4 oligonucleotide generates an EagI restriction site 3* to the NT-4 gene.
  • the PCR generated fragment was digested with EagI and cloned into MscI-EagI digested pRG91.
  • the resulting plasmid, pRG173, consisted of the LamB signal sequence fused to the mature region of hNT-4 (the glycine at amino acid residue 81 in the HG7-2 translated sequence) under the transcriptional control of the lacUVS and T7 ⁇ l.l promoters and the translational control of the T7 ⁇ l.l ribosome binding site.
  • This plasmid also possessed the ropl deletion which increases plasmid copy number.
  • the construction was confirmed by restriction enzyme analysis of purified plasmid DNA, DNA sequence analysis of the purified plasmid, in vitro synthesis of a protein of the approximate size estimated to be encoded by pRG173, and in vivo synthesis of a protein of the appropriate size estimated to be encoded by pRG173 and possessing neurite outgrowth stimulating activity (see discussion, supra) , and having the appropriate N-terminus as determined by amino acid sequencing (GVSETAPAE [SEQ ID NO:25]).
  • the soluble fraction was dialyzed against 50 mM Tris-HCl, pH 8.5 at 4°C for 5 hrs then diluted 10-fold with 20 mM MES, pH 6.0 and loaded on a Fast S-Sepharose column equilibrated with 20 mM MES, pH 6.0 and eluted with 1 M NaCl in 20 mM MES, pH 6.0.
  • Recombinant human.NT-4 protein that stimulated E8 DRG outgrowth was recovered in the 1 M NaCl wash.
  • the insoluble fraction was resuspended and homogenized in 83 ml 8 M guanidinium-HCl, 50 mM Tris-HCl, pH 8.5, 10 mM NaCl, ImM EDTA.
  • the insoluble fraction was dialyzed against 7 M urea, 50 mM Tris-Cl, pH 8.5, and loaded onto a Fast DEAE-Sepharose column equilibrated in 7 M urea, 50 mM Tris-Cl, pH 8.5.
  • the breakthrough fractions were collected and dialyzed against 7 M urea, 100 mM histidine, pH 5.5, 1 mM EDTA and loaded on a Fast S-Sepharose column equilibrated in 7 M urea, 100 mM histidine, pH 5.5, 1 mM EDTA, hNT-4 was eluted with a 0-1M NaCl gradient in the same buffer. Fractions containing hNT-4 were pooled and dialyzed against 100 mM histidine, pH 5.5, 1 mM EDTA. Approximately 95% of the hNT-4 protein fractionated with the insoluble material.
  • SUBSTITUTE SHEET were sacrificed by carbon dioxide asphyxiation, and embryos were rapidly removed and placed in ice-cold medium for further dissection.
  • Spinal cords were removed aseptically from rat embryos of 14 days gestation. The spinal cord was severed caudal to the bulb (at the level of the first dorsal root ganglion) , freed of sensory ganglia and adhering meninges. The cord was then subdivided into ventral and mediodorsal segments for separate cultures. The ventral spinal cord tissues were diced into small pieces and incubated in 0.1% trypsin (GIBCO) and 0.01% deoxyribonuclease type 1 (Sigma) in PBS at 37°C for 20 minutes.
  • GEBCO trypsin
  • Sigma deoxyribonuclease type 1
  • Trypsin solution was then removed, rinsed and replaced with medium consisting of 45% Eagle's minimum essential medium (MEM), 45% Ham's nutrient mixture F12 (F12) , 5% heat inactivated fetal calf serum (GIBCO) , 5% heat inactivated horse serum (GIBCO) , glutamine (2 mM) , penicillin G (0.5 U/ml) , and streptomycin (0.5 ug/ l) .
  • MEM Eagle's minimum essential medium
  • F12 45% Ham's nutrient mixture F12
  • F12 5% heat inactivated fetal calf serum
  • GBCO 5% heat inactivated horse serum
  • glutamine (2 mM) penicillin G
  • streptomycin 0.5 ug/ l
  • the filtered cell suspension were then subjected to a modification of the fraction procedure described by Schnaar and Schaffner (1981, J. Neurosci. 1:204-217). All steps were carried out at 4°C. Metrizamide was dissolved in F12:MEM (1:1) medium, and a discontinuous gradient was established which consisted of a 18% metrizamide cushion (0.5 ml), 3ml of 17% metrizamide, 3ml of 12% metrizamide, and 3ml of 8% metrizamide was prepared.
  • the filtered ventral spinal cord cell suspension (2.5ml) obtained as described above was layered over the step gradient, the tube was centrifuged at 2500g for 15 minutes using a swing-out rotor (Sorvall HB4) . Centrifugation resulted in three layers of cells: fraction I (at 0-8% interface) , fraction II (at 8-12% interface) , and fraction III (at 12-17% interface) .
  • SUBSTITUTESHEET each interface were removed in a small volume (about lml) , rinsed twice with serum-free defined medium consisting of 50% F12 and 50% MEM, supplemented with glutamine (2 mM) , insulin (5 ug/ml) , transferrin (lOOug/ l) , progesterone (20 nM) , putrescine (100 uM) , and sodium selenite (30nM) (Bottenstein and Sato, 1979, PNAS 76:514-517). Viable cell count was obtained by hemocytometer counting in the presence of trypan blue.
  • Fractionated ventral spinal cord cells (enriched with motorneurons) were then plated at a density of 100,000 cells/cm 2 in 6 mm wells precoated with poly-L-ornithine (Sigma: 10 ⁇ g/ml) and laminin (GIBCO: 10 ⁇ g/ml) . Treatment with NT-4 was given on the day of plating. Cultures were maintained in serum-free defined medium at 37°C in 95% air/5% C0 atmosphere at nearly 100% relative humidity. On day 2 (48 hours) , cells were harvested for measurements of choline acetyltransferase (CAT; Fonnum, 1975, J. Neurochem. 24:407-409.
  • CAT choline acetyltransferase
  • the insoluble fraction was assayed on E8 dorsal root ganglia (DRG) explants and showed no bioactivity.
  • the recombinant human NT-4 protein recovered from the 1 m NaCl wash of the soluble fraction was assayed for activity in dissociated motor neuron cultures prepared as described supra at Example Section 9.3, and in other assays.
  • Addition of recombinant human NT-4 at a 1:20 dilution resulted in a 3.6 fold increase in choline acetyltransferase activity in the motor neuron enriched culture 48 hours after treatment.
  • the 3.6 fold increase was measured in relation to untreated (C-NT) and buffer (C- Buffer) controls ( Figure 14) .
  • the ability to express a biologically active form of human NT-4 in an in vitro prokaryotic expression system substantially increases the ease at which the production of human recombinant NT-4, peptides or derivatives thereof may be scaled up for both therapeutic and diagnostic applications.
  • the present example thus teaches that a DNA sequence encoding a human NT-4 is amenable to transcription and translation in a prokaryotic system such that human NT-4 is expressed and the biologically active form is amenable to purification schemes such that the activity remains subsequent to purification.
  • HG7-2 (human NT-4 genomic clone) 75070
  • the present invention is not to be limited in scope by the deposited microorganisms or the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

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Abstract

Procédé de production et de récupération d'une neurotrophine recombinée. Il consiste (1) à cultiver une cellule hôte non animale transformée à l'aide d'une molécule d'ADN recombiné comportant une séquence régulatrice d'expression liée activement à une séquence d'ADN codant une neurotrophine; (2) à dissoudre la neurotrophine dans une solution comportant une quantité suffisante d'un inhibiteur de protéase pour inhiber la dégradation de la neurotrophine par les protéases, et un agent de dénaturation puissant, ladite solution étant essentiellement exempte d'agents de réduction; (3) à substituer à l'agent de dénaturation puissant un agent de dénaturation faible; (4) à régler la solution de sorte qu'elle comprenne un acide aminé de base dont la concentration est suffisante pour maintenir la solubilité de la neurotrophine dans un milieu non dénaturant; (5) à retirer l'agent de dénaturation faible; et (6) à purifier la neurotrophine. Dans un mode spécifique de réalisation, on décrit également la production bactérienne de neurotrophine-4 recombinée.
EP92914222A 1991-06-12 1992-06-11 Production and recovery of recombinant neurotrophins Withdrawn EP0590059A4 (en)

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PL191400B1 (pl) 1996-11-15 2006-05-31 Genentech Inc Sposób izolowania neurotrofiny z mieszaniny białek i kompozycja neurotrofiny
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CA2111110A1 (fr) 1992-12-23
EP0590059A4 (en) 1995-09-06
JPH06508036A (ja) 1994-09-14
NZ243084A (en) 1995-08-28
AU2238792A (en) 1993-01-12
IE921888A1 (en) 1992-12-16
WO1992022665A1 (fr) 1992-12-23
PT100580A (pt) 1993-09-30
IL102170A0 (en) 1993-01-14

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