Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E. coli.
The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains. We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector. A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E. coli. Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 or Met475. We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains. JM105 harboring pKF13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium. The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 micrograms of FXIII A-chains per liter of culture. The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast. These results document that FXIII A-chains can be readily expressed and purified from E. coli culture and that they retained properties similar to those of purified human factor XIII A-chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Lai
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TS
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Santiago
M A
MA
Achyuthan
K E
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Greenberg
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CS
D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli; D005176:Factor XIII / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005337:Fibrin / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D005176:Factor XIII; D011503:Transglutaminases; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases