Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae

• Published in: Protein expression and purification Vol. 3; no. 3; p. 185
• Main Authors: Przysiecki, C T, Joyce, J G, Keller, P M, Markus, H Z, Carty, C E, Hagopian, A, Sardana, M K, Dunwiddie, C T, Ellis, R W, Miller, W J
• Format: Journal Article
• Language: English
• Published: United States 01-06-1992
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Factor V - antagonists & inhibitors; Factor X - antagonists & inhibitors; Factor Xa; Genes, Synthetic; Humans; Invertebrate Hormones - biosynthesis; Invertebrate Hormones - genetics; Invertebrate Hormones - isolation & purification; Invertebrate Hormones - secretion; Leeches - chemistry; Leeches - genetics; Mating Factor; Molecular Sequence Data; Peptides - genetics; Promoter Regions, Genetic; Protein Sorting Signals - genetics; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - secretion; Saccharomyces cerevisiae - metabolism
• ISSN: 1046-5928
• DOI: 10.1016/1046-5928(92)90014-N

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.