[go: up one dir, main page]

WO1989009818A1 - Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2 - Google Patents

Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2 Download PDF

Info

Publication number
WO1989009818A1
WO1989009818A1 PCT/US1989/001418 US8901418W WO8909818A1 WO 1989009818 A1 WO1989009818 A1 WO 1989009818A1 US 8901418 W US8901418 W US 8901418W WO 8909818 A1 WO8909818 A1 WO 8909818A1
Authority
WO
WIPO (PCT)
Prior art keywords
phospholipase
pla
polypeptide
human
dna
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.)
Ceased
Application number
PCT/US1989/001418
Other languages
English (en)
Inventor
Ruth M. Kramer
R. Blake Pepinsky
Catherine Hession
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen Inc
Original Assignee
Biogen Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Biogen Inc filed Critical Biogen Inc
Publication of WO1989009818A1 publication Critical patent/WO1989009818A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to processes for purifying acid stable phospholipase A 2 .
  • the invention relates to processes for the purification of phospholipase A 2 from biological sources, such as non-pancreatic human sources which contain very small amounts of that enzyme. More specifically, this invention relates to the purification and characterization of phospholipase A 2 from human platelets and from human rheumatoid synovial fluid.
  • This invention also relates to polypeptides corresponding to at least a portion of the amino terminal amino acid sequence of human platelet and rheumatoid synovial fluid phospholipase A 2 , and antibodies thereto, as well as antibodies to purified, intact, acid-stable phospholipase A 2 for use in the treatment or diagnosis of inflammation and tissue injury associated with various diseases.
  • This invention further relates to DNA sequences which encode human inflammatory phospholipase A 2 and methods for producing phospholipase A 2 in hosts transformed with recombinant DNA molecules comprising those DNA sequences.
  • Phospholipases A 2 (phosphatide 2-acylhydrolase, EC 3.1.1.4, FLA 2 ) are a family of lipolytic proteins that specifically cleave the acyl ester linkage at the sn-2 position of glycerophospholipids., These enzymes are ubiquitous and are present in virtually every cell type from bacteria to man. Nearly all of the phospholipases A 2 studied to date have a molecular weight of between 10 and 15 kilodaltons, but they differ substantially in amino acid sequence.
  • Secretory phospholipases A 2 may be divided into two categories: digestive (produced and secreted by digestive organs, such as the pancreas), and inflammatory (produced and secreted by inflammatory cells, such as platelets or neutrophils, or found in inflammatory fluids, such as rheumatoid synovial fluid).
  • digestive organs such as the pancreas
  • inflammatory cells such as platelets or neutrophils, or found in inflammatory fluids, such as rheumatoid synovial fluid.
  • phospholipase A 2 In mammals, phospholipase A 2 is found in abundant quantities in the pancreas. Other cellular and extracellular mammalian phospholipases A 2 are found in much smaller amounts.
  • non-pancreatic phospholipases A 2 have been found in seminal plasma, synovial fluid, septic shock serum, and bronchoalveolar lavage fluid of alveolar proteinosis (P. Vadas and W.
  • Pruzanski "Biology of Disease. Role of Secretory Phospholipases A 2 in the Pathobiology of Disease", Lab. Invest., 55, pp. 391-404 (1986)). Most of the m.ammalian phospholipases A 2 are acid-stable and all are are calcium-dependent to varying degrees. To date, with one exception, non-pancreatic phospholipases A 2 demonstrate no immunological cross-reactivity with pancreatic phospholipase A 2 (J. G. N. DeJung et al. "Monoclonal Antibodies against an Intracellular Phospholipase A 2 from Rat Liver and their Cross-Reactivity with Other Phospholipases A 2 ", Eur. J. Biochem., 164, pp. 129-35 (1987)).
  • Intracellular phospholipases A 2 are involved in various physiological functions, including membrane phospholipid turnover, repair of membrane peroxidation damage, transmembrane signaling, cell membrane dynamics and generation of lipid mediators.
  • the control and regulation of phospholipases A» is complex and involves many factors, including free calcium concentration, molecular entities involved in transmembrane signaling, and the physiochemical state of the phospholipid substrate (H. van den Bosch in Comprehensive Biochemistry, vol. 4, pp. 313-57, J. N. Hawthorne arid G. B. Ansell, eds., Elsevier Amsterdam (1982)).
  • Phospholipases A 2 are also known to mediate a variety of pathophysiological conditions through the products of protein catalysis - - lysophospholipids and arachidonic acid.(J. Chang et al., "Phospholipase A 2 : Function and Pharmacological Regulation", Biochem. Pharmacol., 36, pp. 2429-36 (1987); P. Vadas and W. Pruzanski, Lab. Invest., 55, pp. 391-404 (1986); A. A. Farooqui et al., "Phospholipases, Lysophospholipases, and Lipases and Their Involvement in Various Diseases", Neurochem. Path., 7, pp. 99-128 (1987)).
  • Lysophospholipids are fusogenic and cytotoxic. Subsequent metabolism of phospholipase A 2 catalytic products by certain protein cascades leads to several potent, biologically active substances. These include prostaglandins, hydroxylated fatty acids, leukotrienes and platelet activating factor, all of which have been implicated in inflammation or hypersensitivity, or both.
  • phospholipases A 2 play important roles in inflammation and tissue injury associated with various diseases, such as viral and bacterial infections, skin and connective tissue diseases, such as psoriasis, gastrointestinal disorders, such as pancreatitis and ulcers, ischemias, myocardial infarction, atherosclerosis, pulmonary dysfunctions, such as asthma, acute respiratory distress syndrome and alveolar proteinosis, septic shock, thrombosis, multiple sclerosis, demyelinating diseases and rheumatoid arthritis.
  • diseases such as viral and bacterial infections, skin and connective tissue diseases, such as psoriasis, gastrointestinal disorders, such as pancreatitis and ulcers, ischemias, myocardial infarction, atherosclerosis, pulmonary dysfunctions, such as asthma, acute respiratory distress syndrome and alveolar proteinosis, septic shock, thrombosis, multiple sclerosis, demyelinating diseases and rheumatoid arthritis.
  • Pancreas-derived phospholipase A 2 has been purified, sequenced and structurally defined (H. M. Verheij et al., "Structure and Function of Phospholipase A 2 ", Rev. Physiol. Biochem. Pharmacol. , 91, pp. 91-203 (1981)).
  • the protein is produced in the form of an inactive precursor which is stored in secretory grctnules. Once secreted in the intestine the precursor is activated by limited tryptic proteolysis, leading to the formation of the active phospholipase and a small polypeptide. No evidence for such a precursor has been obtained with respect to inflammatory phospholipases A 2 .
  • phospholipase A 2 from rabbit inflammatory peritoneal exudate (S. Forst et al., "Structural and Functional Properties of a Phospholipase A 2 Purified from an Inflammatory Exudate", Biochemistry, 25, pp. 8381-85 (1986)), phospholipase A 2 from secreted rat platelets (M.
  • the present invention solves the problems referred to above by providing processes for purifying acid stable phospholipases A 2 from biological sources, such as mammalian cells and extracellular fluid, plant cells, insect cells, yeast and other fungi, and bacteria. Specifically, these processes permit the purification of inflammatory phospholipases A 2 to homogeneity from human, non-pancreatic sources such as platelets and rheumatoid synovial fluid.
  • the phospholipases A 2 purified from human platelets and rheumatoid synovial fluid by the processes of this invention are characterized by a common amino-terminal amino acid structure, which differs significantly from that of pancreaticderived phospholipase A 2 .
  • phospholipase A 2 purified from rheumatoid synovial fluid exhibits an inflammatory activity that is at least 100-fold greater than that of the pancreatic-derived protein in an assay of inflammatory action that measures formation of paw edema in the rat after subplant or injection of purified phospholipase A 2 (S. Brain et al, "Action of Phospholipase A on Mast Cell Histamine Release and Paw Edema in the Rat", Brit. J. Pharmacol., 59, pp. 440-41 (1977)).
  • This invention also relates to the production of polypeptides corresponding to at least a portion of the amino acid sequence of these inflammatory phospholipases A 2 .
  • polypeptides as well as antibodies to these polypeptides and antibodies to intact, purified phospholipases A 2 , are useful for pharmacological, therapeutic and diagnostic purposes.
  • DNA sequences encoding all or part of the deduced amino acid sequence of these phospholipases A 2 are useful as diagnostics for the evaluation and monitoring of diseases, or as probes for the isolation of cDNA or genomic clones coding for human inflammatory phospholipases A 2 .
  • This invention als'o relates to the production of phospholipase A 2 using recombinant techniques.
  • DNA sequences coding for the phospholipases A 2 of this invention recombinant DNA molecules characterized by those sequences and unicellular hosts transformed with those molecules are employed to produce phospholipases A 2 by culture of those transformed hosts.
  • Figure 1 depicts the elution profile of a partially purified preparation of human platelet phospholipase A 2 off of a cation exchange chromatography column.
  • Figure 2 depicts the elution profile of a partially purified preparation of human platelet phospholipase A 2 off of a gel filtration column.
  • Figure 3, panel A depicts the elution profile of a . partially purified preparation of human platelet phospholipase A 2 off of a reverse phase HPLC column.
  • Figure 3, panel B depicts the corres- ponding phospholipase A 2 activity of the eluted fractions.
  • Figure 4 depicts in tabular form the purification of phospholipase A 2 from isolated human platelets.
  • Figure 5 depicts an Immobilon blot of the human platelet phospholipase A 2 -containing fractions collected following HPLC.
  • Figure 6 depicts the elution profile of a partially purified preparation of human rheumatoid synovial fluid phospholipase A 2 off of a cation exchange chromatography column.
  • Figure 7 depicts the elution profile of a partially purified preparation of human rheumatoid synovial fluid phospholipase A 2 off of a gel filtration column.
  • FIG 8 panel A, depicts the elution profile of a partially purified preparation of human rheumatoid synovial fluid phospholipase A 2 off of a reverse phase HPLC column.
  • Figure 8, panel B depicts the corresponding protein activity of the eluted fractions.
  • Figure 9 depicts in tabular form the purification of phospholipase A 2 from human rheumatoid synovial fluid
  • Figure 10 depicts an Immobilon blot of the human rheumatoid synovial fluid phospholipase A 2 -containing fractions collected following HPLC.
  • Figure 11A depicts portions of sequencing plasmid pNN01.
  • Figure 11B depicts the restriction map of PSQ 130.
  • Figure 12 depicts the DNA sequence of 6 .2 kb HindI I I fragment of PLA 2 8.5 EMBL3 and the ammno acid sequence of human inflammatory phospholipase A 2 derived therefrom.
  • Figure 13 depicts a comparison of the amino acid sequences of bovine pancreatic phospholipase A 2 , phospholipase A 2 from C. atrox venom, and the derived amino acid sequence from the 6.2 kb HindIII fragment of PLA 2 8.5 EHBL3.
  • Figure 14A depicts schematically the synthesis of BG368 from BG312.
  • Figure 14B depicts the restriction map of PLA 2 6.2 BG368 3(+).
  • Figure 14C depicts the restriction map of PLA 2 3.8 BG341(+).
  • Figure 14D depicts the restriction map of BG341.
  • Figure 15A depicts a restriction map of pJODS.
  • Figure 15B depicts a restriction map of PLA 2 3.8 JODS.
  • This invention relates to processes for isolating and purifying acid stable phospholipases A 2 from biological sources.
  • phospholipases A 2 may be purified to homogeneity as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino-terminal amino acid sequence analysis.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • This invention also relates to polypeptides which are characterized by at least a portion of the amino terminal 19 amino acids of human inflammatory phospholipase A 2 purified from platelets and rheumatoid synovial fluid and to the DNA sequences which encode them.
  • one embodiment of the process of this invention begins with the step of acidextracting the protein from a biological source, such as sonicated platelets or rheumatoid synovial fluid.
  • the acid in the extract is then exchanged for a buffer suitable for cation exchange chromatography and the preparation contacted with a cation exchange resin.
  • the phospholipase A 2 is then eluted from the resin, concentrated and further purified away from dissimilar molecular weight contaminants by molecular sizing. Fractions containing phospholipase A 2 activity are purified to homogeneity by reverse-phase HPLC.
  • the process of this invention further comprises the step of electrophoresing said phospholipase A 2 and transferring said electrophoresed phospholipase A 2 to a solid support.
  • the process comprises the further initial step of extracting or releasing the protein from the cell.
  • This may be achieved by any of a number of well-known lysing techniques, such as sonication, homogenization, French press, chemical lysis or enzymatic lysis.
  • Mechanical lysis techniques are preferable, because they do not introduce any extraneous proteins or organic chemicals into the phospholipase A 2 preparation. The most preferred method of lysis is sonication.
  • Acid extraction of the protein according to this invention may be achieved with any acid of any concentration having a pH below about 4.5.
  • the acid is a mineral acid, such as hydrochloric acid, phosphoric acid or sulfuric acid and has a pH or about 1.0.
  • the most preferable acid is sulfuric acid at a concentration of about 0.18 N, which gives a pH of about 1.
  • the protein In order to perform cation exchange chromatography on the phospholipase A 2 preparation, the protein should be in a buffer that is compatible with the cation exchange resin. Numerous methods of achieving buffer exchange are known in the art, including dialysis, ultrafiltration and desalting. Because the phospholipase A 2 is a small protein of about 13,000 daltons, dialysis or ultrafiltration must be carried out using a membrane of suitably small pore size. The most preferred method of buffer exchange is dialysis using a membrane that has a molecular weight cutoff of about 3500 daltons. Various cation exchange resins that are commercially available may be used in the processes of this invention.
  • Examples of some of these resins are Fast S, Mono S, CM-Sepharose, SP-Sepharose and phosphate-cellulose.
  • the preferred properties of the resin used in the process of this invention are high flow rate, the ability to bind phospholipase A 2 at the cation concentration of initial contact and the ability to release phospholipase A 2 under higher ionic conditions.
  • Cation exchange may be performed batchwise, or preferably in a column.
  • the most preferable conditions for cation exchange are initially binding the protein to a Fast S column in 200 mM NaCl, 50 mM acetate, pH 4.5 and eluting the protein with a linear gradient of 200 mM - 2 M NaCl. Using these conditions, the protein will elute at about 1 M NaCl.
  • Active fractions from cation exchange may be concentrated by any standard technique, prefer ⁇ ably one which does not concurrently concentrate ions.
  • the most preferable means of concentration is ultrafiltration using a membrane with a low molecular weight cutoff, such as a YM 5 membrane (Amicon).
  • a membrane with a low molecular weight cutoff such as a YM 5 membrane (Amicon).
  • Any one of a number of commercially available molecular sizing chromatography resins may be employed in the processes of this invention.
  • the resin will be such that the phospholipase A 2 will elute in the included volume. In this manner, the majority of higher molecular weight contaminants will be removed by elution in the void volume of the column.
  • the preferred molecular sizing resins are Biogel P30, Biogel P60, Sephadex G-25, Sephadex G-50, Sephadex G-75 and Utragel AcA54. The most preferred resin is Sephadex G-50 superfine.
  • Active fractions are further purified by reverse phase HPLC.
  • Any hydrophobic resin that is compatible with HPLC may be used with the process of this invention.
  • preferred resins are C18, C8, C4, C3, and phenyl.
  • the most preferred resin is C4.
  • This invention also relates to phospholipases A 2 produced according to the above described processes.
  • Phospholipase A 2 is most preferably characterized by enzymatic activity. For example, phospholipase A, cleaves and releases into the assay supernatant [ 3 H]-oleic acid from autoclaved, [ 3 H]oleic acid-labelled E. coli.
  • Other phospholipase A 2 substrates include, but are not limited to, phosphatidylcholine and phosphatidylethanolamine.
  • phospholipase A 2 may be characterized by its reaction with a specific anti body in assays well-known in the art such as ELISA, Western Blots and immunoprecipitation.
  • Phospholipase A 2 purified according to this invention may be used to raise monoclonal or polyclonal antibodies. It may also be cleaved with various endo- and exopeptidases to produce the polypeptides of this invention. As demonstrated in the following examples, such purified protein was used as a source of amino acid sequence data to permit the synthesis of specific polypeptides which elicit site-specific anti-phospholipase A 2 antibodies. The amino acid sequence data was then employed to obtain nucleotide probes useful in isolating and selecting a DNA sequence encoding phospholipase A 2 from a genomic or cDNA library.
  • the amino acid sequence of phospholipase A 2 purified by the processes described above may be obtained by directly sequencing the material recovered from reverse phase HPLC according to this invention. More preferably, and according to an alternate embodiment of the present invention, the phospholipase A 2 is first subjected to discontinuous SDS-polyacrylamide gel electrophoresis (U. K. Laemmli, "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature,
  • the solid support should be compatible with a protein sequencer.
  • Preferred solid supports are activated glass filters or, more preferably, polyvinylidene difluoride ("PVDF") membranes.
  • PVDF polyvinylidene difluoride
  • the transfer of electrophorejsed phospholipase A 2 to the support may be achieved by capillary transfer, or more preferably, by electrophoretic transfer.
  • proteins may be visualized with any of a number of well-known protein stains, such as Coomassie Blue R-250.
  • the major phospholipase A 2 band is excised from the membrane and subjected to protein sequencing.
  • Protein sequencing may be achieved by standard techniques, preferably using automated Edman degradation, such as with an Applied Biosystems 470A gas phase sequencer.
  • the amino terminal 19 amino acids of both human rheumatoid synovial fluid phospholipase A 2 and human platelet phospholipase A 2 purified according to this invention are both characterized by the amino acid sequence: H 2 N-Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu.
  • Such 100% homol ⁇ gy s ⁇ ggests that other human inflammatory phospholipases A 2 may also contain this sequence.
  • This amino terminal sequence differs from that of any phospholipase A 2 that has been purified previously from either human or non-human sources. Due to limitations in the number of amino acids that can be accurately sequenced by standard techniques, the entire amino acid sequence of phospholipase A 2 is most preferably derived from the nucleotide sequence of a full-length human inflammatory phospholipase A 2 DNA or cDNA clone. The amino acid sequence obtained from the purified natural product has been used to confirm the identification of phospholipase A 2 clones isolated according to the processes of the present invention. This invention also relates to polypeptides which correspond in amino acid sequence to at least a portion of the amino terminal 19 amino acids of human inflammatory phospholipase A 2 .
  • polypeptides may be used to immunize animals and raise specific antibodies.
  • Antibodies to small, weakly immunogenic polypeptides may be elicited by crosslinking the polypeptide to a carrier prior to injec tion into an animal.
  • carrier molecules include, but are not limited to, keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) and cytochrome c.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Methods for crosslinking are also well known in the art and include the use of bifunctional cross-linking reagents, such as glutaraldehyde.
  • Such antibodies are useful in humans and other mammals as anti-inflammatory therapeutics and disease modifying agents in diseases where pathogenic phospholipase A 2 has been implicated.
  • the methods of treatment and their dosage levels and requirements are well recognized in the art and they may be chosen by those of skill in the art from available methods and techniques.
  • the antibodies may be combined with a pharmaceutically acceptable adjuvant for administration to a patient in an amount effective to provide anti-inflammatory effects and accordingly to lessen the severity and course of symptoms.
  • the dosage and treatment regimens will depend upon factors such as the patient's health status, the severity and course of symptoms and the judgment of the treating physician.
  • compositions characterized by antibodies to phospholipase A 2 include, viral and bacterial infections, skin and connective tissue diseases, such as psoriasis, gastrointestinal disorders, such as pancreatitis and ulcers, ischemias, myocardial infarction, atherosclerosis, pulmonary dysfunctions, such as asthma, acute respiratory distress syndrome and alveolar proteinosis, septic shock, thrombosis, multiple sclerosis, demyelinating diseases and rheumatoid arthritis.
  • These antibodies may also be employed as diagnostics in determining phospholipase A 2 levels in tissues, body fluids, inflammatory cells and other cells using any conventional immunoassay technique.
  • Phospholipase A 2 purified by the process of this invention, or recombinantly made, may be used in drug screening programs designed to search for inhibitors that can be used as anti-inflammatory, anti-arthritic and anti-thrombotic agents.
  • the present invention also relates to DNA sequences which encode all or a portion of the amino terminal 19 amino acids of human inflammatory phospholipase A 2 .
  • DNA sequences are preferably synthesized as a combination of oligonucleotides to account for the degeneracy of the genetic code.
  • These DNA sequences, individually or in combination, are useful as probes to permit the isolation and selection of DNA sequences coding for intact phospholipase A 2 and phospholipase A 2 -like polypeptides from various DNA and cDNA libraries, the synthesis of which is well-known in the art.
  • Such DNA sequences may be employed to produce large amounts of phospholipase A 2 , substantially free from other proteins of human origin.
  • libraries include chromosomal gene banks and cDNA or DNA libraries prepared from tissue or cell lines that are demonstrated to produce phospholipase A 2 . These cell lines, as well as techniques for constructing DNA and cDNA libraries, are well known in the art.
  • DNA sequences of the present invention are also useful to probe phospholipase A 2 mRNA levels in inflammatory cells (e.g., neutrophils, monocytes, lymphocytes) and many other cells (e.g., synoviocytes, endothelial cells, smooth muscle cells).
  • inflammatory cells e.g., neutrophils, monocytes, lymphocytes
  • many other cells e.g., synoviocytes, endothelial cells, smooth muscle cells.
  • phospholipase A 2 -like polypeptides are defined as poly peptides which 1) are recognized by antibodies to native phospholipase A 2 in any standard immunoassay, or 2) will elicit antibodies which recognize native phospholipase A 2 in any standard immunoassay, or 3) demonstrate phospholipase A 2 enzymatic activity. It should be understood that a variety of cloning and selection techniques might theoretically be useful in locating and identifying DNA or cDNA sequences of this invention that encode phospholipase A 2 other than the hybridization of oligonucleotides to genomic clones illustrated in the following examples. [See e.g., T.
  • Partial or full-length DNA or cDNA sequences may be used in appropriate recombinant DNA molecules to transform appropriate eukaryotic and prokaryotic hosts for the production of the phospholipase A 2 and phospholipase A 2 -like polypeptides encoded by them.
  • the DNA sequences and recombinant DNA molecules of the present invention may be expressed using a wide variety of host/vector combinations.
  • useful vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from
  • E.coli including col El, pCRl, pBR322, pMB9 and RP4, phage DNAs, e.g., the numerous derivatives of ⁇ phage, e.g., NM 989, and other DNA phages, e.g., M13 and other filamentous single-stranded DNA phages, vectors useful in yeasts , such as the 2 ⁇ plasmid, vectors useful in animal cells, such as those containing SV-40, adenovirus and retrovirus derived DNA sequences (e.g., BG368 and BG341) and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other derivatives thereof.
  • phage DNAs e.g., the numerous derivatives of ⁇ phage, e.g., NM 989, and other DNA phages, e.g., M13 and other filamentous single-strande
  • Such expression vectors are also characterized by at least one expression control sequence that may be operatively linked to the phospholipase A 2 DNA sequence inserted in the vector in order to control and to reflate the expression of that cloned DNA sequence.
  • useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage ⁇ ., the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40, the major late promoter of adenovirus, and other sequences known to control the expression of genes of prokaryotic
  • vectors that enable the expression of the cloned phospholipase A 2 ⁇ related DNA sequences in eukaryotic hosts, such as animal and human cells [e.g., P. J. Southern and P. Berg, J. Mol. Appl. Genet., 1, pp. 327-41 (1982); S. Subramani et al., Mol. Cell. Biol., 1, pp. 854-64 (1981); R. J. Kaufmann and P. A. Sharp, "Amplification And Expression Of Sequences Cotransfected with A Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol Biol., 159, pp. 601-21 (1982); R. J. Kaufmann and P. A.
  • an expression vector useful in this invention need not have a restriction endonuclease site for insertion of the chosen DNA fragment. Instead, the vector could be joined to the fragment by alternative means.
  • the expression vector, and in particular the site chosen therein for insertion of a selected DNA fragment and its operative linking therein to an expression control sequence is determined by a variety of factors, e.g., number of sites susceptible to a particular restriction enzyme, size of the protein to be expressed, susceptibility of the desired protein to proteolytic degradation by host cell enzymes, contamination or binding of the protein to be expressed by host cell proteins difficult to remove during purification, expression characteristics, such as the location of start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art.
  • the choice of a vector and an insertion site for a DNA sequence is determined by a balance of these factors, not all selections being equally effective for a given case.
  • Useful expression hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, such as E.coli SG-936, E.coli HB 101, E.coli W3110, E.coli X1776, E.coli X2282, E.coli MC1061, E.coli DHI, and E.coli MRCl, Pseudomonas, Bacillus, such as Bacillus subtilis, Streptomyces, yeasts and other fungi, animal cells, such as COS cells and CHO cells, human cells, insect cells and plant cells in tissue culture.
  • E.coli such as E.coli SG-936, E.coli HB 101, E.coli W3110, E.coli X1776, E.coli X2282, E.coli MC1061, E.coli DHI, and E.coli MRCl
  • Pseudomonas Bacillus, such as Bacillus subtilis, Streptomyces, yeasts and other fungi
  • animal cells
  • host/expression vector combinations function with equal efficiency in expressing the DNA sequences of this invention or in producing the phospholipase A 2 -like polypeptides.
  • a particular selection of a host-expression . vector combination may be made by those of skill in the art after due consideration of the principles set forth herein without departing from the scope of this invention. For example, the selection should be based on a balancing of a number of factors.
  • genomic clone contains phospholipase A 2 expression control sequences, such as promoters, ribosome binding sites, and polyadenylation signals in addition to the phospholipase A 2 coding sequence
  • expression vectors may be unnecessary.
  • the genomic clone alone may be used to transfect eukaryotic hosts, which, in turn can express phospholipase A 2 .
  • Such hosts include cells well-known in the art, such as mouse L-cells or CHO cells.
  • the present invention provides three different mehtods in which to produce recombinant phospholipase A 2 -like polypeptides.
  • These methods include heterologous promoter-mediated expression of phospholipase A 2 cDNAs, heterologous promoter-regulated expression of phospholipase A 2 genomic DNA, and native promoter-mediated expression of phospholipase A 2 genomic DNA.
  • the latter two methods are only applicable to eukaryotic cells that are able to perform proper splicing; out of introns.
  • the present invention also relates to DNA sequences which hybridize to the foregoing DNA sequences, as well as DNA sequences which, due to the degeneracy of the genetic code, code on expression for human phospholipase A 2 -like polypeptides coded for on expression by the foregoing DNA or cDNA sequences.
  • Figure 1 shows that the phospholipase A 2 eluted with approximately 1 M NaCl at fractions 74-82 - We pooled these fractions and concentrated them to 0.8 ml using an Amicon ultrafiltration stirred cell with a YM 5 membrane.
  • the pooled peak fractions from the gel filtration column (6 ml; 100 ⁇ g protein) were further purified on a C4 reverse-phase HPLC column
  • FIG 3 panel B, demonstrates that approximately 35% of the applied phospholipase A 2 activity was recovered in a single peak contained in fraction 45.
  • Figure 3, panel A indicates the activity eluted at about 32% acetonitrile.
  • Figure 4 depicts, in tabular form, the entire purification process.
  • the final yield of phospholipase A 2 from platelets was 34% and the protein was purified over 1,100,000-fold over the starting material.
  • the total phospholipase A 2 activity increased 63-fold over that observed in the sonicate and was assumed to be 100%.
  • the purification-fold was estimated assuming 100% recovery of enzymatic activity during these steps.
  • Synovial fluid was aspirated from patients diagnosed with classical rheumatoid arthritis, as defined by American Rheumatism Association criteria. We removed ceils and debris from synovial fluids by centrifugation at 4°C for 20 min at 3,000xg in a Sorvall RC3B centrifuge. The synovial fluids were stored at -70°C before further use.
  • the pooled peak fractions from the gel filtration column (6 ml; 100 ⁇ g protein) were further purified on a C4 reverse-phase HPLC column (Vydac; 0.46 ⁇ 25 cm) that was equilibrated at 29°C with 0.1% trifluoroacetic acid (TFA).
  • the column was then developed at a flow rate of 1 ml/min with a 45 minute gradient (0-75% acetonitrile in 0.1% TFA), collecting 0.5 ml fractions.
  • the column eluate was monitored at 214 nm (AUF 0.2) and 280 nm (AUF 0.05).
  • Figure 9 demonstrates, in tabular form, the entire purification process.
  • the final yield of phospholipase A 2 from rheumatoid synovial fluid was 57% and the protein was purified over 100, 000-fold over the starting material.
  • a unit of activity for human platelet phospholipase A 2 was defined as the amount of protein necessary to release 1 ⁇ 10 6 cpm of [ 3 H]-oleate in 15 min at 37°C.
  • the polypeptide was coupled to keyhole limpet hemocyanin with glutaraldehyde before being used to immunize rabbits.
  • 1.5 mg of the p ⁇ lypeptide-KLH complex was emulsified with Freund's complete adjuvant and administered intramuscularly.
  • 0.75 mg of the polypeptide-KLH complex was emulsified with Freund's incomplete adjuvant and administered intramuscularly. Animals were bled every 2 weeks and sera assayed for anti-human inflammatory phospholipase A 2 titer by ELISA.
  • phospholipase A 2 -like polypeptides as well as mature phospholipase A 2 may be similarly synthesized and used to raise anti-human inflammatory phospholipase A 2 antibodies. Additionally, phospholipase A 2 -like polypeptides of sufficient size and immunogenicity may be used directly to elicit antibodies to phospholipase A 2 without coupling to KLH.
  • nucleotide More than one nucleotide was introduced at various positions during the synthesis of these oligonucleotides, to account for the degeneracy of the genetic code.
  • the product of any single oligonucleotide synthesis was actually a mixture of oligonucleotides, all of which potentially coded for the corresponding hexapeptide.
  • the following code is used to designate nucleotides: A- adenine N- adenine, thymidine, guanidine, or cytidine T- thymidine
  • PLA 2 -06 5' ATC ATPu CGPu TGPu AAPu TT 3'
  • PLA 2 -07 5' ATC ATPy CGPu TGPu AAPu TT 3'
  • PLA 2 -08 5' ATC ATPy CTPu TGPu AAPu TT 3'
  • PLA 2 -09 5' GTN GTPy AAPy TTZ ATC AT 3'
  • PLA 2 -10 5' GTN GTPu AGPy TTZ ATC AT 3'
  • PLA 2 -11 5' GTN GTPy AGPy TTZ ATC AT 3'
  • oligonucleotides based on the hexapeptide Thr-Thr-Gly-Lys-Glu-Ala were synthesized: PLA 2 -12: 5' GCPy TCPy TTPu CCPu GTPu GT 3' PLA 2 -13: 5' GCPy TCPy TTPy CCPy GTPy GT 3'
  • oligonucleotides were synthesized on an Applied Biosystems 380A automated DNA synthesizer, using the procedure described by L. J. McBride and M. H. Caruthers, "The Synthesis of Oligodeoxypyrimidines on a Polymer Support", Tetrahedron Letters, 24, pp. 245-48 (1983).
  • Recombinant bacteriophage were selected by plating on E.coli MP801 cells (a gift of Dr. Mark Pasek, Biogen Inc., Cambridge, MA), a P2 lysogen of SG4119.
  • the fragment was cloned into HindIII-digested pNN01 that had been treated with calf intes- tinal alkaline phosphatase.
  • the novel synthetic portion of pNN01 is shown in Figure 11A.
  • a BamHI-Ncol fragment of the 6.2 kb PLA 2 insert which hybridized to the PLA 2 oligonucleotide probes of this invention, had the following nucleotide sequence: C CAT GGG AAT TTG GTG AAT TTC CAC AGA ATG ATC AAG TTG ACG ACA GGA AAG GAA GCC GCA CTC AGT TAT GGC TTC TAC GGC TGC CAC TGT GGC GTG GGT GGC AGA GGA TCC. This sequence encodes the polypeptide:
  • the entire DNA sequence of the 6.2 kb PLA 2 insert as well as the amino acid sequence coded for in the exons is shown in Figure 12. Potential intron splice sites are indicated by arrowheads.
  • the coding sequence for mature phospholipase A 2 begins at nucleotide 2722 (arrow, Figure 12) and is contained within exon 2.
  • Exon 2 begins at nucleotide 2702 and encodes 6 in-frame amino acids preceding the amino terminal asparagine residue of mature phospholipase A 2 . We believe that these 6 amino acids encode the carboxy terminal 6 amino acids of the phospholipase A 2 signal sequence.
  • nucleotides 2453 and 2492 An open reading frame of 14 amino acids beginning with a methionine residue and having characteristic properties of a signal sequence is located between nucleotides 2453 and 2492. This nucleotide sequence also terminates with a characteristic GT splice site at nucleotides 2493-2494. It is unlikely that this represents the N-terminal portion of the in vivo signal, because no promoter-like sequences are found within the 100 nucleotides located 5' to this region.
  • Exons 3 and 4 which encode the remainder of phospholipase A 2 and an in-frame stop codon, are located at nucleotides 3105-3211 and 5383-5523, respectively.
  • the putative polyadenylation signal, AATAAA is located at nucleotides 5771-5776 (underscored in Figure 12 ) .
  • the mature FLA 2 polypeptide coded for by exons 2, 3 and 4 consists of 124 amino acids and has the formula: NLVNFHRMIK LTTGKEAALS YGFYGCHCGV GGRGSPKDAT DRCCVTHDCC YKRLEKRGCG TKFLSYKFSN SGSRITCAKQ DSCRSQLCEC DKAAATCFAR NKTTYNKKYQ YYSNKHCRGS TPRC.
  • amino acids are represented by single letter codes as follows:
  • the 3 exons of the genomic clone contained within the 6.2 kb PLA 2 EMBL3 8.5 HindIII fragment encode phospholipase A 2 ) based on the following observations.
  • the clone encodes the identical N-terminal amino acid sequence identified for the purified native enzyme by protein sequencing (see Example 4). This sequence represents an amphiphilic alpha-helix that is typical for all phospholipases A 2 sequenced to date. It also encodes the highly conserved lipophilic residues within this alpha-helix (e.g., Leu 2 , Phe 5 and Ile 9 ).
  • the clone codes for a cluster of basic amino acids (e.g., Arg 7 , Lys 11 and Lys 15 ) which is believed to be an important determinant in the interaction of phospholipase A 2 with specific biological targets.
  • the clone encodes a characteristic stretch of amino acids Tyr 25 -Gly-Cys-XCys-Gly-X-Gly-Gly-X-X-Pro 37 and Asp 49 , where X is any amino acid, that are part of the calcium binding loop of phospholipases A 2 .
  • the clone encodes the characteristic amino acid residues that constitute the active site of all phospholipases A 2 , namely His 48 , Asp 99 , Tyr 52 and Tyr 73 .
  • the phospholipase A 2 amino acid sequence coded for by the 6.2 kb HindIII fragment of genomic DNA clone PLA 2 EMBL3 8.5 also exhibits the placement of half-cysteine residues that is typical for group II phospholipases A 2 . Thus, it contains a cysteine residue at amino acid 50 and has an extension of several amino acid residues at the C-terminus which ends in a half cysteine.
  • Plasmid PLA 2 6.2 BG368 3(+) ( Figure 14B) was synthesized as follows: PSQ 130 was digested with HindIII and the 6.2 kb PLA 2 insert was isolated by preparative gel electrophoresis through low-melt agarose. The gel slice containing this fragment was excised and stored at 4°C.
  • the animal cell expression parent vector, BG368, was constructed as follows: As depicted in Figure 14A, we cut animal cell expression vector BG312 [R. Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp.
  • BG368 was linearized by digestion at the unique HindIII site in the polylinker region.
  • BG368 contains the SV40 origin of replication and enhancer sequence, the adenovirus major late promoter, a polylinker region containing unique restriction sites for the insertion of DNA sequences for expression, the SV40 3' untranslated region, including the polyadenylation signal and the 3' splice site.
  • the 3.8 kb PLA 2 insert contains a potential open reading frame encoding an initiating methionine as well as amino acids which are characteristic of signal sequences. This fragment also contains Exons 2, 3, and 4, as well as a donor sequence for splicing the amino terminus of the signal sequence to Exon 2.
  • Plasmid BG341 ( Figure 14D) was also derived from BG312 (R. Cate et al., Cell, 4-5, pp. 685-98 (1986)). After partial digestion of BG312 with Bglll to linearize it at the Bglll site located at approximately position 99, we filled in the Bglll site with Klenow fragment of E.coli DNA polymerase and deoxyribonucleotides then religated the vector. We linearized the vector by partial digestion with BamHI at the site following the SV40 poly A region, filled in the site with Klenow and deoxyribonucleotides and religated the vector.
  • the resultant vector with unique Bglll and BamHI sites was linearized with SmaI and a linker having the sequence 5'GCGGCCGCGCTCGAGCTCG3' was ligated into the SmaI site, thus destroying it and creating a NotI site in the cloning region.
  • BG341 was then linearized by digestion at its unique NotI site.
  • the linearized plasmid was then alkaline phosphatase treated and purified in an identical manner to that described above for BG368.
  • Example 10 Because the transfected COS-7 cells described in Example 10 expressed phospholipase A 2 only transiently, we next constructed cell lines that stably expressed the polypeptide. We employed the commercially available expression host CHO
  • DHFR- the dihydrofolate reductase deletion mutant of the Chinese Hamster Ovary cell line.
  • PLA 2 3.8 JODS Figure 15B
  • PLA 2 3.8 JODS Figure 15A
  • PLA 2 3.8 BG341(+) was also digested with the same enzymes. Both digests were purified on a low melt agarose gel and the appropriate band excised from the gel.
  • the AatII-Notl fragment from PLA 2 3.8 BG341(+) (containing the promoter elements from BG341 as well as the 3.8 kb PLA 2 insert) was ligated to the AatII-Notl fragment of pJODS (containing the DHFR sequence).
  • pJODS containing the DHFR sequence
  • We named the plasmid PLA 2 3.8 JODS We then used this plasmid to transform E.coli JA221 cells and prepared CsCl-banded plasmid DNA for use in animal cell transfection.
  • PLA 2 3.8 JODS was linearized with Aatll. We routinely used 20 ⁇ g of linearized plasmid to transfect CHO DHFR- cells. Transfections, growth of cells and phospholipase A 2 assays were performed as described above.
  • the bacteriophage PLA 2 8.5 EMBL3 contained approximately 16 kb of human DNA, including the PLA 2 insert which encodes mature inflammatory PLA 2 .
  • This phage is modified by standard techniques so that it contains a selectable marker for animal cell expression, such as herpes simplex virus thymidine kinase.
  • Phage particle transfection of mouse L tk- cells is accomplished by the methods of M. Ishiura et al., "Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells", Mol. Cell Biol., 2, pp. 607-16 (1982).
  • L tk - cells are plated in 100 mm2 tissue culture dishes at a density of 5 ⁇ 10 5 - 1 ⁇ 10 6 cells per plate.
  • 1 ml of 3 ⁇ 10 7 pfu/ml phage particles are coprecipitated with calcium phosphate, pH 6.85, at 25°C for 10 minutes.
  • the precipitate is then absorbed on the L cells for 24 hours at 37°C in 5% CO 2 in air.
  • the cells are then washed with Hepes buffered saline, re-fed with ⁇ -MEM and grown for 40 hours.
  • the media is then replaced with HAT media and the cells maintained until clones appear (about 10 days). Tk clones are assayed for phospholipase A 2 activity.
  • DNA mediated transfection can be carried out.
  • DNA is prepared from either PLA 2 8.5 EMBL3 or PSQ 130.
  • the 6.2 kb PLA 2 insert is isolated and purified by digestion with HindIII and preparative gel electrophoresis.
  • PLA 2 8.5 EMBL3 DNA is linearized by digestion with SalI.
  • Each DNA preparation may then used together with an appropriate selectable marker (e.g., DHFR for CHO DHFR- cells, thymidine kinase for L tk- cells) to cotransfect animal cells.
  • DNA-mediated transfection is accoasplished by calcium phosphate precipitation or electroporation (G.
  • BG341 (as a control) were used to prepare total RNA.
  • the transfected cells were lysed in guanidinium isothiocyanate buffer and total RNA prepared by the method of J. M. Chirgwin et al., "Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease", Biochemistry, 18, pp. 5294-99 (1979).
  • OxanI is the equivalent isoschizomer of commercially available restriction enzymes MstIl and Bsu36. All of these enzymes recognize the nucleotide sequence: CCTNAGG. These enzymes cut PSQ 130 at nucleotides 2054-2060 and 3413-3419.
  • the probe hybridized to an 1100 nucleotide mRNA in cells transfected with PLA 2 3.8 BG341(+).
  • This size is consistent with expected transcription from the adenovirus late promoter and predicted splicing and polyadenylation.
  • RNA isolated from cell lines transfected with PLA 2 3.8 BG341(+) or any other PLA 2 genomic clone which expresses human inflammatory phospholipase A 2 by the above method may be used to obtain a PLA 2 cDNA.
  • Poly A + RNA is isolated from total RNA using oligo dT cellulose chromatography.
  • the poly A+ RNA (5-10 ⁇ g) is resuspended in H 2 O at a concentration of 5 ⁇ g/ ⁇ l and treated with 2.5 mM CH 3 HgOH at room temperature for 10 minutes. ⁇ -mercaptoethanol is then added to a final concentration of 0.035 M.
  • cDNA is carried out using a cDNA synthesis kit (BRL; catalogue #8267SA) according to manufacturer's directions.
  • the double stranded cDNA is then ligated to linker 35-36: 5'AATTCGAGCTCGAGCGCGGCCGC3' 3' GCTCGAGCACGCGCCGGCG5' using standard procedures.
  • the cDNA is then size selected using Select 4L, 5L, or 6L columns (5 Prime-3 Prime Inc., West Chester, PA) according to manufacturer's directions.
  • the size-selected cDNA is then ligated to EcoRI digested ⁇ gt10. Aliquots of the ligation reaction are packaged in Gigapack (Stratagene, San Diego, CA) according to the manufacturer's protocol.
  • PLA 2 cDNA clones are then obtained by screening the resultant library with antisense oligonucleotides from the PLA 2 coding region of PLA 2 3.8 BG341(+).
  • PLA 2 -specific mRNA is enriched initially by hybrid selection with PLA 2 genomic DNA using any one of the methods described in R. Jagus, "Hybrid Selection of mRNA and Hybrid Arrest of Translation” Meth. Enzymol., 152, pp. 567-72 (1987).
  • the predicted nucleotide sequence of the PLA 2 coding region of the above-described cDNA is: AAT TTG GTG AAT TTC CAC AGA ATG ATC AAG TTG ACG ACA GGA AAG GAA GCC GCA CTC AGT TAT GGC TTC TAC GGC TGC CAC TGT GGC GTG GGT GGC AGA GGA TCC CCC AAG GAT GCA ACG GAT CGC TGC TGT GTC ACT CAT GAC TGT TGC TAC AAA CGT CTG GAG AAA CGT GGA TGT GGC ACC AAA TTT CTG AGC TAC AAG TTT AGC AAC TCG GGG AGC AGA ATC ACC TGT GCA AAA CAG GAC TCC TGC AGA AGT CAA CTG TGT GAG TGT GAT - AAG GCT GCT GCC ACC TGT TTT GCT AGA AAC AAG ACG ACC TAC AAT AAA AAG TAC CAG TAC TCC AAT AAA AAT AAA A
  • Microorganisms and recombinant DNA molecules prepared by the processes of this invention are exemplified by cultures deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland. These include cultures deposited on July 11, 1988 and identified as:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Rheumatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des procédés de purification de la phospholipase A2 stable aux acides. D'une manière spécifique, l'invention concerne des procédés de purification de la phospholipase A2 à partir de sources biologiques telles que des sources humaines non pancréatiques qui contiennent de très petites quantités de cette enzyme. Plus spécifiquement, cette invention concerne la purification et la caractérisation de la phospholipase A2 à partir de plaquettes sanguines humaines et à partir de fluide synovial rhumatoïde humain. Cette invention concerne également des polypeptides correspondant à au moins une partie de la séquence d'acides aminés à terminaison amino de la phospholipase A2 du fluide synovial rhumatoïde et des plaquettes humains et leurs anticorps, ainsi que des anticorps contre la phospholipase A2 purifiée, stable à l'acide, utilisée pour le traitement ou le diagnostic d'inflammations ou de blessures de tissus associées à diverses maladies. Cette invention concerne également des séquences d'ADN qui codent ces polypeptides. Cette invention concerne aussi des procédés de production de phospholipase A2 dans des hôtes transformés avec des molécules d'ADN recombinant comprenant ces séquences ADN.
PCT/US1989/001418 1988-04-15 1989-04-11 Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2 Ceased WO1989009818A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US18189388A 1988-04-15 1988-04-15
US181,893 1988-04-15
US21949188A 1988-07-12 1988-07-12
US219,491 1988-07-12

Publications (1)

Publication Number Publication Date
WO1989009818A1 true WO1989009818A1 (fr) 1989-10-19

Family

ID=26877609

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1989/001418 Ceased WO1989009818A1 (fr) 1988-04-15 1989-04-11 Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2

Country Status (3)

Country Link
JP (1) JPH03503843A (fr)
AU (1) AU3548289A (fr)
WO (1) WO1989009818A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0435725A1 (fr) * 1989-12-15 1991-07-03 Asahi Kasei Kogyo Kabushiki Kaisha ADN avec information génétique pour la phospholipase D et son utilisation
EP0446350A4 (en) * 1987-12-24 1991-09-25 Teijin Limited Novel human phospholipase a2 and its fragment peptide
EP0476849A3 (en) * 1990-08-24 1992-06-17 Eli Lilly And Company Human cytosolic phospholipase a2
EP0509719A1 (fr) * 1991-04-17 1992-10-21 Eli Lilly And Company Composés, vecteurs et procédés d'expression de la phospholipase A2 cytosolique humaine
WO1993007872A1 (fr) * 1991-10-17 1993-04-29 The University Of Nottingham Inhibiteurs d'enzymes lysosomes pour le traitement de maladies neurodegeneratives
WO1995000649A1 (fr) * 1993-06-25 1995-01-05 Smithkline Beecham Plc Phospholipase a2 associee a une lipoproteine, inhibiteurs de cette phospholipase et son utilisation a des fins diagnostiques et therapeutiques
WO1998055504A1 (fr) * 1997-06-05 1998-12-10 Institut Pasteur PEPTIDES A BASE DE hsPLA2 DU GROUPE II PRODUISANT UN EFFET ANTICOAGULANT
WO1999050278A1 (fr) * 1998-03-31 1999-10-07 Genzyme Corporation Compositions et methodes d'identification de cellules de tumeur du poumon
CN111303298A (zh) * 2020-02-19 2020-06-19 李瑛� 含有磷酸酶的融合蛋白及其产品和应用

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Biochemistry, vol. 25, 1986, American Chemical Society, S Forst et al.: "Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate", pages 8381-8385 *
Chemical Abstracts, vol. 103, no. 1, 8 July 1985, (Columbus, Ohio, US), M.M. Rakhimov et al.: "Biospecific adsorption chromatography of phospholipase A2 from different sources", see page 245 *
Chemical Abstracts, vol. 103, no. 11, 16 September 1985, (Columbus, Ohio, US), T.L. Hazlett et al.: "Affinity chromatography of phospholipase A2 from Naja naja naja (Indian cobra) venom", see page 272 *
Chemical Abstracts, vol. 105, no. 23, 8 December 1986, (Columbus, Ohio, US), R.M. Kramer et al.: "Solubilization and properties of calcium-dependent human platelet phospholipase A2", see page 262 *
Chemical Abstracts, vol. 92, no. 9, 3 March 1980, (Columbus, Ohio, US), J. Salak et al.: "Isolation of corynebacterial enzymes from cultivation media by gel filtration and ion-exchange chromatography", see page 313 *
J. Biochem., vol. 101, no. 5, 1987, M. Hayakawa et al.: "Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A21", pages 1311-1314 *
J. Biochem., vol. 102, no. 1, 1987, H.W. Chang et al.: "Purification and characterization of extra-cellular phospholipase A2 from peritoneal cavity of caseinate-treated rat", pages 147-154 *
J. Biochem., vol. 99, no. 3, 1986, O. Ohara et al.: "Dog and rat pancreatic phospholipase A2: Complete amino acid sequences deduced from complementary DNAs", pages 733-739 *
The Journal of Biological Chemistry, vol. 261, no. 23, 15 August 1986, The American Society of Biological Chemists, Inc., (US), L.A. Loeb et al.: "Identification and purification of sheep platelet phospholipase A2 isoforms", pages 10467-10470 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0446350A4 (en) * 1987-12-24 1991-09-25 Teijin Limited Novel human phospholipase a2 and its fragment peptide
EP0435725A1 (fr) * 1989-12-15 1991-07-03 Asahi Kasei Kogyo Kabushiki Kaisha ADN avec information génétique pour la phospholipase D et son utilisation
EP0476849A3 (en) * 1990-08-24 1992-06-17 Eli Lilly And Company Human cytosolic phospholipase a2
EP0509719A1 (fr) * 1991-04-17 1992-10-21 Eli Lilly And Company Composés, vecteurs et procédés d'expression de la phospholipase A2 cytosolique humaine
US5328842A (en) * 1991-04-17 1994-07-12 Eli Lilly And Company Compounds, vectors and methods for expressing human, cytosolic phospholipase A2
WO1993007872A1 (fr) * 1991-10-17 1993-04-29 The University Of Nottingham Inhibiteurs d'enzymes lysosomes pour le traitement de maladies neurodegeneratives
US5981252A (en) * 1993-06-25 1999-11-09 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7045329B2 (en) 1993-06-25 2006-05-16 Smithkline Beecham Plc Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US8088886B2 (en) 1993-06-25 2012-01-03 Smith-Kline Beecham P.L.C. Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US5968818A (en) * 1993-06-25 1999-10-19 Smithkline Beecham Corporation Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
WO1995000649A1 (fr) * 1993-06-25 1995-01-05 Smithkline Beecham Plc Phospholipase a2 associee a une lipoproteine, inhibiteurs de cette phospholipase et son utilisation a des fins diagnostiques et therapeutiques
US6177257B1 (en) 1993-06-25 2001-01-23 Smithkline Beechman Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US6369045B1 (en) 1993-06-25 2002-04-09 Smithkline Beecham P.L.C. Phospholipase A2 inhibitors thereof and use of same in diagnosis and therapy
US7416853B2 (en) 1993-06-25 2008-08-26 Smithkline Beecham P.L.C. Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7314742B2 (en) 1993-06-25 2008-01-01 Smithkline Beecham Corporation Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7217535B2 (en) 1993-06-25 2007-05-15 Smithkline Beecham P.L.C. Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
US7052862B2 (en) 1993-06-25 2006-05-30 Smithkline Beecham Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy
WO1998055504A1 (fr) * 1997-06-05 1998-12-10 Institut Pasteur PEPTIDES A BASE DE hsPLA2 DU GROUPE II PRODUISANT UN EFFET ANTICOAGULANT
AU757478C (en) * 1998-03-31 2004-03-25 Genzyme Corporation Compositions and methods for the identification of lung tumor cells
AU757478B2 (en) * 1998-03-31 2003-02-20 Genzyme Corporation Compositions and methods for the identification of lung tumor cells
WO1999050278A1 (fr) * 1998-03-31 1999-10-07 Genzyme Corporation Compositions et methodes d'identification de cellules de tumeur du poumon
CN111303298A (zh) * 2020-02-19 2020-06-19 李瑛� 含有磷酸酶的融合蛋白及其产品和应用
CN111303298B (zh) * 2020-02-19 2023-03-21 李瑛� 含有磷酸酶的融合蛋白及其产品和应用

Also Published As

Publication number Publication date
JPH03503843A (ja) 1991-08-29
AU3548289A (en) 1989-11-03

Similar Documents

Publication Publication Date Title
EP0253455A1 (fr) Méthode de préparation de protéines à activité de facteur VIII par utilisation de cellules microbiennes hôtes; vecteurs d'expression, cellules hôtes, anticorps
US5726147A (en) Human mutant tissue factor compositions useful as tissue factor antagonists
JP2515468B2 (ja) ヒト抗トロンビンｉｉｉ
EP1538202B1 (fr) Production de l'alpha-galactosidase A humaine
DE69427997T2 (de) Hybrider mensch/tier faktor viii
EP0335243A2 (fr) Angiogénine mutante humaine (facteur d'angiogénèse avec une activité d'angiogénine supérieure), gènes pour celle-ci et méthode d'expression
EP0275305A4 (fr) Sequences adn pour facteur viii:c modifie et polypeptides semblables au facteur viii:c modifie et procedes de production en grandes quantites de ces polypeptides.
JP2004256541A (ja) インスリン様成長因子結合タンパク質とその組成物
JPS63301797A (ja) 組織因子タンパク質を製造するためのデオキシリボ核酸
JP2000212098A (ja) ヒト・アポリポ蛋白質ａｉおよびａｉ―ミラノを含有する医薬組成物
JP2757987B2 (ja) ヒト抗‐炎症ホスホリパーゼ阻害蛋白
Ghildyal et al. Lack of expression of the tryptase mouse mast cell protease 7 in mast cells of the C57BL/6J mouse.
JP2983224B2 (ja) 哺乳動物の１４―β―ｇａ１レクチン類
AU665034B2 (en) Production of human parathyroid hormone from microorganisms
WO1990006365A1 (fr) Proteines recombinantes presentant des activites d'adipsine et de complement d
WO1989009818A1 (fr) Procede de purification de phospholipase a2 et de production de polypeptides semblables a la phospholipase a2
DE3855080T2 (de) Synoviale phospholipasen
US5622843A (en) Phospholipid transfer proteins and DNA encoding them
US5994296A (en) Human mutant tissue factor compositions useful as tissue factor antagonists
US5405946A (en) Recombinant protein S variants deficient in C4BP binding activity, compositions and therapeutic methods
EP0446931A1 (fr) Facteur neuronotrophique de cil humain, séquence d'ADN encodant le facteur, et production du facteur par technologie ecombinante
EP0677107B1 (fr) Inhibiteur de thrombine provenant de salive de protostomiens
US4950646A (en) DNA sequences, recombinant DNA molecules and processes for producing human lipocortin-like polypeptides
JPH08511157A (ja) Ｘａファクター阻害活性を有する新規ポリペプチド
US6015685A (en) Ancrod proteins, their preparation and use

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1989905319

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1989905319

Country of ref document: EP