KR890014125A - Biologically active protein - Google Patents

Abstract

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Description

Biologically active protein

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1 shows INFα INH active cross section of Sephacryl S-200 gel filtered urine. Column fractions (1 ml) were tested at 1:10 dilution for rhTNFα (1.0 ng / ml) cytotoxicity assay in the presence of actinomycin D (1.0 μg / ml) (oo). Line (-) represents OD ₂₈₀ nm of fraction. The bar is actinomycin D ( ) And urinary actinomycin D plus hrTNFα ( Cell lysis measured by die uptake in response to). Molecular weight markers include dextran blue (DB) for bovine serum albumin (BSA), egg albumin (OA), α-chymotrypsinogen (αCT), ribonuclease A (RNase A) and phenol red (Ф-red) to be.

2 shows the TNFα INH activity cross section of chromatofocusing urine on one-P column. Column fractions (1 ml) were tested at 1:10 dilution for rhTNFα (0.2 ng / ml) cytotoxicity assay in the presence of actinomycin D (1.0 μg / ml) (oo). The line (-) represents the OD ₂₈₀ of the fractions, (......) represents their pH, and the bars are as described in FIG. 3 shows the reversibility of TNFα INH activity. Ring opening (oo) shows OD ₅₇₀ nm measured in the presence of actinomycin D, TNFα INH and rhTNFα. Closed (-) indicates OD ₅₇₀ nm measured in the presence of actinomycin and rhTNFα. ) Represents OD 570n in the presence of actinomycin D (1.0 μg / ml) only.

Claims (25)

Tumor company factor (TNF) A protein that inhibits α-mediated activity and does not inhibit other proteins that covalently have a specific TNF but not all biological activities of TNF. A protein that selectively inhibits tumor company factor α-mediated activity and has one or more of the following characteristics, (a) molecular sieve determined by molecular sieve chromatography, and (b) measured by chromatofocusing method, 5.5 Inhibition of fractional cytotoxicity by standard TNF analysis on L929 cells of rats treated with isoelectric point (PI), (c) actinomycin D, in the range of ˜6.1. TNFα to U937 cells (monocyte tumor cell line), as demonstrated by (d) inhibition of TNF-induced PGF2 released from human fibroblasts and synovial cells, and (e) inhibition of binding of radially labeled TNFα ( ¹²⁵ I-TNFα). Inhibitors that interfere with the binding of (f) the separation of the pre-formed TNFα: U937 cell complex is promoted by the inhibitor in a temperature dependent manner, (g) the inhibitor does not degrade TNF by proteolytic cleavage, (h) the inhibitor does not inhibit IL-1 receptor-binding activity, ie binding of radiolabeled IL-1 ( ¹²⁵ I-IL-Iα) to murine thymoma subtype EL4-6.1. Proteins that inhibit tumor company factor α-mediated activity and have one or more of the following characteristics: (a) 33 KDa molecular weight as measured by SDS PAGE, (b) isoelectric point (pI) within the range of 5.5 to 6.1 as determined by chromatographic focusing method, ( c) inhibition of fractional cytotoxicity by standard TNF assay on L929 cells of rats treated with actinomycin D (d) inhibition of TNF-induced PGF2 released from human fibroblasts and synovial cells, (e) radiolabeled Inhibitors that interfere with the binding of TNFα to U937 cells (monocyte tumor cell lines), as evidenced by inhibition of binding of TNFα ( ¹²⁵ I-TNFα), (f) isolation of the pre-formed TNFα: U937 cell complex in a temperature dependent manner Promoted by an inhibitor, (g) the inhibitor does not degrade TNF by proteolytic cleavage, (h) the inhibitor inhibits IL-1 receptor-binding activity, ie, radiolabeled IL-1 ( ¹²⁵ I- IL-Iα) Inhibits Binding of Rat Thymoma Subtype EL4-6.1 Not removing. The protein according to claim 2 or 3, which has the characteristics of (a) and (b) and at least one of (c) to (h). The protein according to claim 2 or 3, wherein the protein has the properties of (a) to (h). The protein according to any one of claims 1 to 5, which corresponds to a naturally occurring TNFα inhibitor. The amino terminal amino acid sequence of claim 1, wherein the amino-terminal amino acid sequence is as follows: Asp-Ser-Val-Cys-Pro-Gln-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser Protein with Ile. 8. The amino terminal amino acid sequence of claim 7, wherein the amino-terminal amino acid sequence is Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-Pro-Gln-Cys-Asn-Ser-Ile-Asn-Ser- Protein with Thr-Lys. The resulting sequence according to any one of claims 1 to 8, wherein the amino acid sequence has at least 80% homology with one or more deletion, substitution, insertion, inversion and addition sequences of the parental origin or the other and the parent protein. Proteins that contain and have essentially the same biological properties as proteins. The protein of claim 9 having at least 90% homology of the parent protein. The protein of claim 1, wherein the protein is in substantially homogeneous form. The protein of claim 1, wherein the protein is a recombinant protein. The protein of claim 1, which is in a glycosylated state. The protein of claim 1, which is in a substantially aglycosylated state. The foreign DNA of the nucleotide sequence which codes for the protein as defined in any one of Claims 1-12. A DNA comprising a nucleotide sequence encoding a protein as defined in any one of claims 1 to 12. A recombinant expression vector consisting of DNA according to claim 15. A host cell transformed with the expression vector according to claim 17. A method for producing TNFα INH comprising culturing a cell according to claim 18 and isolating TNFα INH protein. A recombinant protein produced by the method of claim 19. From (a) patients with fever, (b) ammonium sulfate precipitation, (c) anion exchange chromatography, (d) cation exchange chromatography, (e) gel filtration, (f) affinity chromatography and (g) inverse A method of making TNFα INH consisting of steps of face FPLC. A protein, wherein the protein is substantially the same as the protein obtained by the method of claim 21. A medicament comprising a TNFα inhibitor as defined in any one of claims 1 to 14 or 22 or a pharmaceutically acceptable and its pharmaceutically acceptable carrier. The protein as defined in any one of claims 1 to 14 or 22 for treatment. A pharmaceutical formulation for treating a condition associated with excess or non-regulated TNFα production, wherein the medicament is TNFα INH as defined in any one of claims 1 to 14 or 22, or a pharmaceutically acceptable derivative thereof. ※ Note: The disclosure is based on the initial application.