Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae.
Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents. A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin. Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction. rHIR was purified by a process that consisted of two chromatographic steps and decolorization. Total yield for the purification process was 3.6 g, or 41%. This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography. Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA. Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.
Lehman
E D
ED
Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, Pennsylvania 19486.
Joyce
J G
JG
Bailey
F J
FJ
Markus
H Z
HZ
Schultz
L D
LD
Dunwiddie
C T
CT
Jacobson
M A
MA
Miller
W J
WJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D003001:Cloning, Molecular; D015870:Gene Expression; D005813:Genes, Synthetic / Q000235:genetics; D014644:Genetic Variation; D006629:Hirudins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D013917:Thrombin / Q000037:antagonists & inhibitors
D006629:Hirudins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D013917:Thrombin