title: Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E. coli.
abstract: 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)
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: T S
authors.initials: TS
authors.lastname: Lai
authors.forename: M A
authors.initials: MA
authors.lastname: Santiago
authors.forename: K E
authors.initials: KE
authors.lastname: Achyuthan
authors.forename: C S
authors.initials: CS
authors.lastname: Greenberg
mesh_terms: 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
chemical_list: 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