EXPRESSION PLASMID CONTAINING DNA FRAGMENT CODING MUTEIN C245S OF HUMAN TISSUE FACTOR WITH INSEPARABLE C-TERMINAL TAG, BACTERIUM E coli PRODUCING PRECURSOR PROTEIN OF MUTEIN C245S OF HUMAN TISSUE FACTOR WITH INSEPARABLE C-TERMINAL TAG, METHOD FOR PRODUCING RECOMBINANT MUTEIN C245S OF HUMAN TISSUE FACTOR WITH INSEPARABLE C-TERMINAL TAG, RECOMBINANT MUTEIN C245S OF HUMAN TISSUE FACTOR WITH INSEPARABLE C-TERMINAL TAG

摘要

FIELD: medicine.;SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.;EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.;9 cl, 5 dwg, 1 tbl, 7 ex