Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae

Abstract

Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired dimer assembly and decreased stability of naturally recurring R260C mutant A subunit for coagulation factor XIII

“…A yeast expression vector RPOT carrying rF13A cDNA was constructed as previously described (25). In vitro mutagenesis was performed by recombinant polymerase chain reaction (PCR) (26) using oligonucleotide primers: for R260C, 5 0 -ACAGTGGAGCTCCAGG GCGTG-3 0 (5 0 -side, sense), 5 0 -GACCCC-ACACAGCTGACTTTG ATGGGATTC-3 0 (middle, antisense), 5 0 -CAAAGTCAGCTGTGT GGGGTCTGCAATGGT-3 0 (middle, sense) and 5 0 -CATCGCTAT TTTCCTGGGGGG-3 0 (3 0 -side, antisense); for IV-del, 5 0 -TACGTC ATTGATGATGCTGTGTATCTGGAC-3 0 (sense from exon III) and 5 0 -ACAGCATCATCAATGACGTATTCCACCCTG-3 0 (antisense from exon V).…”

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

“…Kinetic studies used ZymoGenetics Inc. (Seattle, WA) (rXIIIA) (19). Synthetic substrates for XIIIa were used as supplied in Berichrom® XIIIa assay kits except when supplemented with the glutamine substrate LGPGQSKVIG from Behring Werke (Marburg, Germany).…”

Type‐2 plasminogen‐activator inhibitor is a substrate for trophoblast transglutaminase and Factor XIIIa

“…As estimated by densitometric scanning of SDSgels, its purity was approximately 65%. The recombinant human placental factor XIIIa, zymogen, expressed in yeast, was a gift from Dr. P. Bishop, Zymogenetics Inc. Seattle (Bishop et al, 1990). Human placental factor XIIIa, (fibrogammin) was a gift from Dr. Eckhard Schuler, Behringwerke, Marburg, Germany.…”