KR970701774A - USE OF A RECOMBINANT INHIBITOR FROM ERYTHRINA CAFFRA FOR PURIFYING SERINE PROTEASES for Purification of Serine Proteases - Google Patents

Abstract

The present invention binds a serine protease to an immobilized polypeptide having the activity of an erythrina capra-derived inhibitor DE-3, removes unbound components from the protein mixture, removes the serine protease from the inhibitor, and dissolves the immobilized inhibitor. A method of purifying a serine protease from a protein mixture by isolating it from a serine protease and isolating the serine protease, wherein the polypeptide is used as a polypeptide which is a prokaryotic or eukaryotic expression product of an exogenous nucleic acid. Such inhibitors are distinguished by improved inactivation and are particularly suitable for the purification of tissue plasminogen activators (t-PA and derivatives).

Description

USE OF A RECOMBINANT INHIBITOR FROM ERYTHRINA CAFFRA FOR PURIFYING SERINE PROTEASES for Purification of Serine Proteases

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Claims (19)

The serine protease is bound to an immobilized polypeptide having the activity of Eritri or capra derived inhibitor DE-3, the unbound components are removed from the protein mixture, the serine protease is removed from the inhibitor, and the immobilized inhibitor is removed from the soluble serine protease. A method of purifying serine proteases from protein mixtures by isolating and isolating serine proteases, wherein the polypeptide is used as a polypeptide, either a prokaryotic or eukaryotic expression product of an exogenous nucleic acid, by an anion exchanger, a cation exchanger or a nickel chelate. Purification method characterized in that the chromatographic purification. The method of claim 1 wherein the serine protease is a plasminogen activator. The method of claim 2, wherein the plasminogen activator is a tissue plasminogen activator or a derivative thereof. The method according to any one of claims 1 to 3, wherein said inhibitor is immobilized on an inert carrier. 5. The exogenous nucleic acid of claim 1, wherein the exogenous nucleic acid is essentially identical to a nucleic acid encoding the same polypeptide within the overlapping range of the nucleotides 9 to 527 of SEQ ID NO: 1 or the genetic code. How to feature. Prokaryotic or eukaryotic expression products of exogenous nucleic acids for affinity-chromatographic purification of serine proteases, and have the activity of Eritrina capra derived inhibitor DE-3, chromatographically purified by anion exchangers, cation exchangers or nickel chelates. Use of Polypeptides. Use of a polypeptide according to claim 6 wherein said serine protease is a plasminogen activator. 8. The use according to claim 7, wherein said plasminogen activator is a tissue plasminogen activator or derivative thereof. Exogenous nucleic acid essentially consistent with a nucleic acid encoding the same polypeptide within the sequence or genotype overlapping sequence of Nucleotides 9 to 527 of SEQ ID NO: 1 in such a manner that the host cell can express the polypeptide under appropriate nutritional conditions. A method for producing a polypeptide having the activity of an Eritri or capra-derived inhibitor DE-3 by culturing a prokaryotic or eukaryotic host cell transformed or transfected, and isolating a desired polypeptide, wherein the polypeptide is directed to trypsin of about 1.07 U / mg. It has a non inhibitory activity, The manufacturing method characterized by the above-mentioned. 10. A method according to claim 9, wherein a nucleic acid encoding a nucleic acid of the sequences of nucleotides 9 to 527 of SEQ ID NO: 1 or identical polypeptides within a genetic code overlapping range is used as said nucleic acid. The method of claim 9 or 10, wherein the host cell is E. A method for producing coli cells. The method of claim 9 or 10, wherein said host cell is a yeast cell or CHO cell. Nucleic acids from nucleotides 9 to 527 of SEQ ID NO: 1 which encode a polypeptide having the activity of an erythrina capra derived inhibitor DE-3. A biologically functional plasmid or viral DNA vector comprising the nucleic acid of claim 13. Prokaryotic or eukaryotic host cells stably transformed or transfected with the DNA vector of claim 14. Exogenous nucleic acid essentially consistent with the sequence of nucleotides 9 to 527 of SEQ ID NO: 1 or the sequence encoding the same polypeptide within the genetic code duplication in such a manner that the host cell can express the desired polypeptide under appropriate nutritional conditions; By culturing transformed or transfected prokaryotic or eukaryotic host cells, isolating the desired polypeptide, and having an Eritri or capra derived inhibitor DE-3 activity, which is ca 1.07 U / mg or less for trypsin Polypeptide having the above non-suppressor activity. The polypeptide of claim 16, wherein the expression is carried out in a prokaryotic host cell and has an amino acid sequence of SEQ ID NO: 2. The polypeptide of claim 16, wherein the expression is carried out in a eukaryotic host cell and has the amino acid sequence of SEQ ID NO: 2 without N-terminal methionine. A nucleic acid encoding the same polypeptide within the sequence of nucleotides 9 to 527 of SEQ ID NO: 1 or in a genetic code duplication in such a manner that the host cell can express the polypeptide under appropriate nutritional conditions. Eritrina through culturing prokaryotic or eukaryotic host cells transformed or transfected with essentially identical nucleic acids, separating polypeptides from the host cells, and chromatographic purification on an anion exchanger, cation exchanger or nickel chelate column. A method for producing a recombinant polypeptide having the activity of a capra derived inhibitor DE-3. ※ Note: The disclosure is based on the initial application.