| Title: | Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae |
| Author(s): | Przysiecki CT; Joyce JG; Keller PM; Markus HZ; Carty CE; Hagopian A; Sardana MK; Dunwiddie CT; Ellis RW; Miller WJ; |
| Address: | "Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, Pennsylvania 19486" |
| DOI: | 10.1016/1046-5928(92)90014-n |
| ISSN/ISBN: | 1046-5928 (Print) 1046-5928 (Linking) |
| Abstract: | "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" |
| Keywords: | "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/genetics/;" |
| Notes: | "MedlinePrzysiecki, 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 eng 1992/06/01 Protein Expr Purif. 1992 Jun; 3(3):185-95. doi: 10.1016/1046-5928(92)90014-n" |
