WO2000020610A1 - Disruption du gene kex1 dans pichia et procedes d'expression proteique complete - Google Patents

Disruption du gene kex1 dans pichia et procedes d'expression proteique complete Download PDF

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WO2000020610A1
WO2000020610A1 PCT/US1999/023351 US9923351W WO0020610A1 WO 2000020610 A1 WO2000020610 A1 WO 2000020610A1 US 9923351 W US9923351 W US 9923351W WO 0020610 A1 WO0020610 A1 WO 0020610A1
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protein
gene
kexl
pichia
disruption
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PCT/US1999/023351
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Thomas Boehm
M. Judah Folkman
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The Children's Medical Center Corporation
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • microorganisms it is possible to modify microorganisms in order to make them produce proteins of interest such as, for example, mammalian proteins, artificial proteins, chimeric proteins, and the like.
  • proteins of interest such as, for example, mammalian proteins, artificial proteins, chimeric proteins, and the like.
  • numerous genetic studies have been performed on the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. More recently, genetic tools have been developed so as to use the yeast Pichia pastoris as host cell for the production of recombinant proteins.
  • Pichia offers many of the benefits of E. coli (high-level expression, easy scale-up, and inexpensive growth) combined with many of the advantages of expression in an eukaryotic system (protein processing, folding, and posttranslational modifications).
  • P. pastoris is a methylotrophic yeast. In the absence of a repressing carbon source, such as glucose, P. pastoris is able to use a metabolic pathway that allows it to utilize methanol as a carbon source.
  • the alcohol oxidase promoter controls expression of alcohol oxidase which catalyzes the first step in methanol metabolism. To overcome the low specific activity of the enzyme, large quantities of alcohol oxidase are produced. Typically greater than 30% of the total soluble protein in methanol-induced cells is alcohol oxidase.
  • the AOX1 promoter has been characterized and incorporated into a series of Pichia expression vectors in order to take advantage of the powerful AOX1 promoter to drive high-level expression of recombinant proteins.
  • P. pastoris is a useful candidate for expression of proteins in sufficient quantities (see the review in Romanos, M.A. et al. ( ⁇ 992)Yeast 8, 423-488). Protein yields of more than 1 gram per liter have been described (Tschopp, J.F. et al. (1987) 5,1305-1308; Paifer, E. et al. ( ⁇ 994)Yeast 10, 1415-1419; Laroche, Y. et al.
  • P. pastoris is easily adaptable to large-scale fermentation for the production of recombinant protein. Expression of recombinant proteins in P. pastoris has been carried out in fermentors as small as 1 liter and as large as 10,000 liters. Moreover, recombinant protein expression in P. pastoris is less expensive than expression in insect or mammalian systems. The growth and expression medium does not require expensive supplements such as fetal bovine serum.
  • the present invention provides methods and compositions for recombinant expression of full length proteins in P. pastoris.
  • the invention provides genetic constructs containing a disruption in the
  • KEXl gene that prevent cleavage of basic amino acids, such as lysine, from the carboxy terminal.
  • FIG. 1A is the amino acid sequence of P. pastoris Kexlp(SEQ ID NO: l). Arrows indicate amino acid residues likely to be important for the catalytic activity of Kexlp. Filled triangles denote potential N-linked glycosylation sites.
  • Figure IB is a comparison of the amino acid sequences of S. cerevisiae (top)(SEQ ID NO:2) and P. pastoris Kexlp (bottom) (SEQ ID NO:3).
  • Figures 2A and 2B illustrates the genomic locus of the wild- type SMD1168 and the mutant kexl::SUC2 strains.
  • Figure 2A is a genomic map around the wild type KEXl gene. The numbers in parentheses correspond to the base pair number in the GenBank file
  • Figure 2B is a genomic map of the kexl::SUC2 disruption strain (ol is oligonucleotides used for verification of successful disruption of the KEXl gene). Detailed Description of the Invention
  • the present invention provides a recombinant nucleic acid construct comprising a disrupted KEXl gene of Pichia that prevents cleavage of one or more basic amino acids from the carboxy terminal of a protein expressed therewith.
  • the Pichia is a Pichia pastoris.
  • the basic amino acid is a lysine.
  • the disruption is a nucleic acid deletion, insertion or addition within the KEXl gene.
  • the present invention also provides a method of expressing a full length protein comprising transfecting a gene encoding the protein into a recombinant nucleic acid construct and promoting the expression of the gene, wherein the recombinant nucleic acid construct comprises a disrupted KEXl gene of Pichia that prevents cleavage of one or more basic amino acids from the carboxy terminal of a protein expressed therewith.
  • the Pichia is a Pichia pastoris.
  • the basic amino acid is a lysine.
  • the disruption is a nucleic acid deletion, insertion or addition within the KEXl gene.
  • the invention also provides a method for expressing a full length protein comprising transfecting a gene encoding the protein into a recombinant nucleic acid construct and promoting the expression of the gene, wherein the recombinant nucleic acid construct comprises a disrupted KEXl gene of Pichia that prevents cleavage of one or more basic amino acids from the carboxy terminal of a protein expressed therewith, and wherein the gene has been modified to contain one or more basic amino acids at the carboxy terminal.
  • the Pichia is a Pichia pastoris.
  • the basic amino acid is a lysine.
  • the disruption is a nucleic acid deletion, insertion, susbstitution or addition within the KEXl gene.
  • a or “an” herein is meant or more than one, depending upon the context.
  • protein is meant any naturally occurring or modified sequence of amino acids, i.e. a polypeptide of any length. Such a protein can be a peptide fragment of a naturally occurring or modified protein, in addition to chimeric, fusion or labelled proteins, for example.
  • the invention provides that a basic amino acid can be a lysine, histadine, or arginine. Under some physiological conditions, basic amino acids can include asparagine and glutamine.
  • Cleavage of such basic residues can be prevented by the present invention whether the basic amino acid is at or near the carboxy terminal of the protein of interest.
  • the invention contemplates that more than one basic amino acid may be located at or near the carboxy terminal of a protein in order to facilitate expression of an increased amount of the full length of the desired protein.
  • the present invention provides a KEXl disruption strain wherein the disruption of the KEXl reading frame allows expression of an increased amount of full length protein with the carboxy terminal intact.
  • the KEXl disruption strain is useful for the full length expression of other proteins with a carboxy terminal, or carboxy terminal region, basic amino acid.
  • protein that can be expressed with the present invention include, but are not limited to, endostatinTM protein, and the anaphylatoxins C3a and C5a. It has been shown that the carboxy terminal arginines of C3a and C5a are necessary for full biological activity.
  • the 3.5 kb P. pastoris KEXl gene locus has been deposited in the GenBank database and is available under the Accession Number AF095574. Any disruption of the KEXl gene that results in the production of full length protein as desired is contemplated by the present invention.
  • Any disruption of the KEXl gene that results in the production of full length protein as desired is contemplated by the present invention.
  • One such example of a disruption involving a deletion and insertion is described below in the examples wherein a portion of the KEXl gene is deleted and replaced with a portion of a Sarcomyces cerevisiae SUC2 gene.
  • the KEXl disruption constructs of the present invention are also useful for producing proteins having a basic amino acid purposefully added to the carboxy terminus in order to protect the carboxy terminus from degradation by other carboxypeptidases.
  • the present invention provides for the inhibition of degradation of recombinant proteins by genetically constructing the protein with a carboxy terminal region lysine, or other basic amino acid(s), that is compatible with protein activity.
  • the KEXl strain can be used to make epitope labeled proteins.
  • epitope tagging of proteins has become useful, because it allows the detection of proteins with highly specific, high-affinity antibodies within several weeks without the need to express a sufficient quantity of protein for injection into rabbits and without the need to depend on adequate antibody titers.
  • the FLAG-tag (Asp-Tyr-Lys-Asp 4 - Lys)(SEQ ID NO:4) is among the frequently used amino acid sequences.
  • the present invention provides methods and genetic constructs which increase the amount of full length protein expressed therewith. In some cases with expression of protein on the KEXl disrupted construct, a mixture of proteins is formed, some having intact carboxy terminii and some not intact. It appears that lysis of yeast cells occurs which releases proteases into the culture-supernatant.
  • the invention provides a means to overcome this problem by using a double deletion strain, such as crossing the KEXl disruption strain with the PRC1 deletion strain (Ohi, H. et al. (1996) Yeast 12, 31-40).
  • Carboxy terminal degradation can also be minimized by a short methanol induction phase.
  • degradation of the carboxy terminal lysine by other enzymes can be minimized using either shaker flask purification or short fermentation runs.
  • intact proteins can easily be separated from degraded forms using cation exchange chromatography.
  • the invention provides that the disrupted KEXl constructs allow for an increased amount of full length protein to be expressed therein.
  • embodiments of the invention provide that the cloning of the disrupted KEXl gene allows construction of an overexpression strain, which may be more efficient in the cleavage of carboxy terminal basic amino acids.
  • This strain is useful during expression of proteins, where proteolytic activation and removal of carboxy terminal basic amino acids is important for the generation of active protein.
  • a classical example of such a protein is insulin. Construction of a P. pastoris strain overexpressing the Kexlp, Kex2p and Stel3p proteins is very efficient in secreting active insulin. Thim et al. (Proc. Natl. Acad. Sci. 83, 6766-6770 (1986)) have reported that in S. cerevisiae the proper processing of insulin is a limiting factor.
  • the preferred SMDl 168 strain of P. pastoris was used.
  • SMDl 138 is a pep4 mutant strain of P. pastoris deficient in proteinase A activity. Proteinase A is required for the proteolytic activation of a number of proteases, including carboxy peptidase Y.
  • SMDl 168 can be used to reduce proteolysis of some recombinant proteins expressed in the strain.
  • the preferred pPIC9K Pichia expression vector also available from Invitrogen, was used. The pPIC9K Pichia expression vector allows one to obtain Pichia strains that contain multiple copies of the gene of interest.
  • the pPIC9K vector carries the kanamycin resistance gene which confers resistance to G418 in Pichia. Spontaneous generation of multiple insertion events, which occur in Pichia at a frequency of 1-10%, can be identified by the level of resistance to G418. Pichia transformants are selected on histidine deficient medium and screened for their level of resistance to G418. An increased level of resistance to G418 indicates multiple copies of the kanamycin resistance gene and of the gene of interest.
  • the pPIC9K vector allows secreted expression of the gene of interest.
  • the pA0815 vector is specially designed to generate multiple copies of the gene of interest in a single vector.
  • the vector contains a Bgl II site upstream of the 5' AOX1 gene and a unique BamH I site downstream of the 3' AOX1 transcription termination (TT) signal.
  • Four steps are performed to generate multiple copies of the gene of interest.
  • the gene is cloned into the unique EcoR I site in the vector.
  • the construct is digested with BamH I and Bgl II to release the "expression cassette" containing the AOX1 promoter, gene of interest, and 3' AOX1 TT.
  • multiple copies of the expression cassette are generated in vitro by ligation.
  • compositions and methods disclosed and claimed herein are useful in other species and strains of Pichia and that other expression systems can be used.
  • Invitrogen also offers a Pichia methanolica expression system, to which the presently disclosed methods are applicable.
  • the invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention.
  • EndostatinTM protein is a potent angiogenesis inhibitor.
  • endostatinTM protein is expressed in Pichia pastoris, analysis of the expressed protein by mass spectrometry indicates that the protein is truncated. N-terminal sequence analysis determined that the N- terminus was intact, suggesting that the C-terminal lysine was missing.
  • Kexlp a carboxypeptidase B-like enzyme, can cleave lysine and arginine residues from the C- terminus of peptides and proteins. It was discovered that the KEXl homologue in P. pastoris is responsible for the loss of the C- terminal lysine of endostatinTM protein.
  • Example 1 Construction of a P. pastoris genomic library The polymerase chain reaction was performed according to standard protocols using either TAQ (Boehringer Mannheim) or PFU polymerase (Stratagene). All PCR fragments were cloned into the plasmid pCRTM2.1, a TA-cloning vector (Invitrogen). PFU amplified fragments were isolated with the PCR-Q1A Quick spin purification kit (Qiagen), incubated for 10 min with TAQ polymerase and ligated into the TA-vector. PFU polymerase was used for the amplification of the KEXl gene locus from genomic
  • PCR analysis of the KEXl disruption strains single colonies were inoculated into 1.5 ml YEPD medium and grown overnight to saturation. The cell pellet was resuspended in 200 ⁇ l of lysis buffer (1% SDS, 2% Triton X-100, 100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA).
  • DNA was isolated from the P. pastoris strain SMDl 168 using the Qiagen Genomic DNA Purification Kit. 2 ⁇ g of genomic DNA were digested for 4 h with 30 Units EcoR l. The digested DNA was repurified with the QIAEX II kit and 100 ng were ligated into the Lambda ZAP II vector according to the protocol supplied by the manufacturer (Stratagene). 3 ⁇ l of the ligation mix were used for packaging the vector into phage particles (Gigapack 111 Gold packaging kit, Stratagene). Eleven million primary clones were obtained and 200,000 clones were amplified. The Qiagen
  • Lambda DNA isolation protocol was used to obtain phage DNA from agarose plate lysates. 200 ng of phage DNA were used for PCR analysis.
  • the murine and human endostatinTM protein cDNAs were amplified using PCR and cloned into the Xhol/Notl sites of the expressing vector pPIC9 (pPIC9-mES and pPIC9-hES, respectively, Invitrogen). This cloning created a fusion protein of the alpha-factor secretion signal with endostatin.
  • Murine endostatinTM protein should be secreted starting with the published N-terminus His-Thr- His-Gln-Glu-Phe and human endostatinTM protein should start with His-Ser-His-Arg-Glu-Phe, if correct processing by the Kex2p and the Stel3p occurs. These constructs were verified by sequencing. A 1.6 kb Bamm/Xbal fragment of pPIC9-mES and pPIC9-hES was subcloned into pPIC9K.
  • the plasmid was transfected into the SMDl 168 strain.
  • This strain has a deletion in the PEP4 gene, which is important for the activation of carboxypeptidase Y (CPYp).
  • CPYp carboxypeptidase Y
  • the SMDl 168 strain should have a lower capacity to degrade proteins from the C- terminus compared to the wild type P. pastoris strain GST115.
  • Murine endostatinTM protein was purified from yeast supernatant grown in shaker flasks. For shaker flask purification, the recommendations of the manufacturer were followed (Invitrogen). Rich medium buffered with potassium phosphate at pH 6.0 was used. The cultures were induced with methanol for 48-
  • ammonium sulfate solution was prepared by dissolving 767 g of ammonium sulfate
  • endostatinTM protein Fractions containing endostatinTM protein were pooled, dialyzed against PBS and filter sterilized. The protein was stored at -20° C. Murine and human endostatinTM protein were also purified from yeast culture-supernatant harvested from a fermenter (5 liter bench-top B. Braun fermenter). Complex medium was used and the fermentation conditions recommended by Invitrogen were followed. The pH was kept constant at 6.5. The pH of the complex medium was kept constant at 6.5, because it has been shown that endostatinTM protein is a zinc-binding protein.
  • zinc- binding may be necessary for full biological activity and it protects the N-terminal amino acids from proteases and therefore endostatinTM protein from inactivation (Boehm et al., submitted).
  • Growing the yeast cells below pH 6.5 may cause dissociation of zinc from the binding pocket and may allow proteases to cleave off some N-terminal amino acids, which would result in the purification of inactive protein.
  • Atomic absorption of endostatinTM protein isolated from shaker flasks and a fermenter showed a 1: 1 ratio of zinc to protein.
  • the methanol induction phase was either 40 h or 70 h.
  • the supernatant was dialyzed against PBS and concentrated. Ammonium sulfate precipitation was not possible and the solution was directly applied onto a heparin-Sepharose column. After washing the column with PBS and 0.25 M NaCl, 20 mM Tris, pH
  • endostatinTM protein was eluted using a 0.25 to 1.5 M NaCl gradient. Fractions containing endostatinTM protein were pooled and dialyzed against PBS. A second chromatography step was necessary to purify endostatinTM protein to apparent homogeneity. A HiPrep 26/60 Sephacryl 3-200 HR gel filtration column (Pharmacia) was used, equilibrated in PBS. EndostatinTM protein containing fractions were filter sterilized and stored at -20° C at a protein concentration of 0.5-2.0 mg/ml, determined using the Bio- Rad Bradford assay with immunoglobulin as the standard protein.
  • EndostatinTM protein was purified to apparent homogeneity from the shaker flasks as shown by SDS PAGE. N-terminal sequence analysis showed the correct N-terminus but mass spectrometry revealed that one amino acid is missing from the C- terminus. The molecular weight determined by mass spectrometry was 20248.4 Da, which is in good agreement with the calculated molecular weight of endostatinTM protein without a C-terminal lysine of 20243.79. The "real" calculated molecular weight of endostatinTM protein without the C-terminal lysine would be 20247.79 but 4 Da have been subtracted because two disulfide bonds are formed. Free cysteines in endostatinTM protein purified from the P.
  • endostatinTM protein in a fermenter required the use of a heparin-Sepharose and a gel-filtration step to purify it to apparent homogeneity. Under the high cell density growth conditions during fermentation a fraction of endostatinTM protein was further degraded. Mass spectrometry analysis indicated that the C-terminal serine exposed by removal of the lysine was removed. The calculated molecular weight of endostatinTM protein without a
  • the KEXl gene encodes a carboxypeptidase B-like enzyme, which is involved in the processing of the killer toxin and the alpha-factor (Dmochowska, A. et al. (1987) Cell 50, 573-584).
  • the Kex2p and the Stel3p cleave off the alpha-factor signal sequence, which allows secretion of alpha-factor or the protein of interest into the culture medium. Because the alpha-factor secretion signal was used for expression of endostatinTM protein, it was hypothesized that the Kexlp homologue in P. pastoris removed the C-terminal lysine.
  • the P. pastoris KEXl gene was cloned and disrupted in order to test this hypothesis.
  • a PCR approach was used to clone the KEXl gene.
  • the S. cerevisiae Kexlp and the CPYp from S. cerevisiae and P. pastoris contain a highly conserved motif around the active serine residue, Gly-Glu-Gly-Ser-Tyr-Ala-Gly (SEQ ID NO:5).
  • Degenerative oligonucleotides of this motif were designed and as a second primer T3 from the vector sequence was used. 200 ng of phage library DNA were used as a template.
  • oligonucleotide combined with a T3 primer from the lambda vector (pBluescript) amplified a 1.8 kb fragment at 62° C annealing temperature, 5'-TG NCC CGC RTA RCT YTC ACC-3'(SEQ ID NO:6). Sequence analysis followed by a BLAST database search showed the highest homology to the S. cerevisiae KEXl gene.
  • the T7 primer from pBluescript and a reverse primer obtained from the sequence of the 1.8 kb PCR fragment were used to obtain a 4.2 kb fragment 3' of the Gly-Glu-Gly-Ser-Tyr-Ala-Gly (SEQ ID NO:5) motif.
  • the P. pastoris Kexlp does not have the very distinct 105 residue stretch rich of aspartic and glutamic acids found in the S. cerevisiae sequence (Fig. IB). Nevertheless, the P. pastoris Kexlp has a short hydrophilic-acidic region upstream of the conserved membrane spanning domain. This transmembrane domain might target the Kexlp to the late Golgi compartment, as has been shown for the S. cerevisiae Kexlp (Cooper, A. et al. (1989) Mol. Cell. Biol. 9, 2706- 2714; Bryant, N.J. et al. (1993) J. Cell Science 106, 815-822). A second hydrophobic region is located at the N-terminus and may serve as a signal sequence (Watson, M.E.E. (1984) Nucl. Acids Res. 12, 5145-5156).
  • the P. pastoris Kexlp has six potential N-linked glycosylation sites (AspXxxSer/Thr) compared to four in the S. cerevisiae homologue (Fig. 1A).
  • the two sites between amino acid 449-469 in the S. cerevisiae Kexlp and 430-440 in P. pastoris Kexlp are conserved (Fig. IB).
  • Example 5 Disruption of the KEXl gene with SUC2
  • major parts of the reading frame were disrupted and the question of whether endostatinTM protein could be purified with the C-terminal lysine was investigated.
  • SUC2 gene from S. cerevisiae as a selectable marker (Figs. 2 A and 2B).
  • the SUC2 locus encodes the invertase gene, which should allow P. pastoris cells to grow efficiently on minimal sucrose plates.
  • P. pastoris cells grow slowly on minimal sucrose (MS) plates.
  • a 2.2 kb fragment of the KEXl locus including the ATG and the stop codon was amplified from SMDl 168 genomic DNA (Fig. 2B) and cloned into the TA-cloning vector pCRTM2.1.
  • the oligonucleotides contained a 5' Smal and 3' SnaBl restriction site.
  • the resulting plasmid (pCRTM2.1-KEXl) was amplified in the methylation deficient strain SCSI 10 (Stratagene) and cut with Clal/Ndel.
  • a 2.9 kb segment of the SUC2 gene was PCR amplified from 5288C genomic DNA with Cla ⁇ and Nde l flanking restriction sites and cloned into the pCRTM2.1-KEXl plasmid.
  • the disruption plasmid (pCRTM2.1-kexl ::SUC2) was digested with
  • Recombinant endostatinTM protein was assayed on a Voyager DE-Elite or Voyager DE-STR BiospectrometryTM workstation (Perceptive Biosy stems).
  • Molecular mass analysis was performed on 2 ⁇ l purified protein in PBS cocrystallized with 2 ⁇ l of a 10 mg/ml sinapinic acid/myoglobin solution (3,5-dimethoxy-4- hydroxy-cinnamic acid in 1:2 v/v acetonitrile/water, 0.1 % TFA and 200 ng/ml horse myoglobin (Sigma) as internal standard). Under these conditions the error of the mass determination is below 0.1%.
  • the isoelectric point of intact murine endostatinTM protein is 8.93. Removal of the C-terminal lysine shifts the isoelectric point to 8.54. This pH difference was exploited to separate intact from degraded endostatinTM protein using cation exchange chromatography.
  • a MonoS HR 5/5 column (Pharmacia) was used to separate intact endostatinTM protein and endostatinTM protein without the C-terminal lysine.
  • 0.1 to 1 mg of purified endostatinTM protein in PBS was loaded onto the column using the FPLC system from Pharmacia. The column was washed with 50 mM HEPES, pH
  • the first peak corresponds to endostatinTM protein without a C-terminal lysine and to a certain extent without serine
  • the second peak was intact endostatinTM protein as determined by mass spectrometry.
  • the percentage ratio of intact to degraded endostatinTM protein was about 52 to 48.
  • Human endostatinTM protein was also expressed in the KEXl gene deletion strain. Most of the protein showed a molecular weight close to the calculated mass of 20091.5 Da (4 Da have been subtracted).

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Abstract

La présente invention concerne des procédés et des compositions permettant l'expression recombinée de protéines complètes dans la levure Pichia. En outre, cette invention concerne des produits de synthèse renfermant une disruption du gène KEX1 empêchant le clivage d'amino-acides basiques, tels que la lysine, à partir de l'extrémité C-terminale des protéines ainsi exprimées.
PCT/US1999/023351 1998-10-07 1999-10-07 Disruption du gene kex1 dans pichia et procedes d'expression proteique complete WO2000020610A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6797488B1 (en) 1997-12-08 2004-09-28 Beth Israel Deaconess Medical Center Methods of producing anti-angiogenic proteins
WO2011064282A1 (fr) * 2009-11-25 2011-06-03 Novo Nordisk A/S Procédé de production de polypeptides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691183A (en) * 1993-07-07 1997-11-25 University Technology Corporation CD4+ T-lymphoctye proteases and genes encoding said proteases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691183A (en) * 1993-07-07 1997-11-25 University Technology Corporation CD4+ T-lymphoctye proteases and genes encoding said proteases

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6797488B1 (en) 1997-12-08 2004-09-28 Beth Israel Deaconess Medical Center Methods of producing anti-angiogenic proteins
WO2011064282A1 (fr) * 2009-11-25 2011-06-03 Novo Nordisk A/S Procédé de production de polypeptides
US8815541B2 (en) 2009-11-25 2014-08-26 Novo Nordisk A/S Method for making polypeptides

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