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Titolo:
Expression and characterization of recombinant human antithrombin III in Pichia pastoris
Autore:
Mochizuki, S; Hamato, N; Hirose, M; Miyano, K; Ohtani, W; Kameyama, S; Kuwae, S; Tokuyama, T; Ohi, H;
Indirizzi:
Welfide Corp, Div Pharmaceut Res, Drug Discovery Labs, Osaka 5731153, Japan Welfide Corp Osaka Japan 5731153 ug Discovery Labs, Osaka 5731153, Japan
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 1, volume: 23, anno: 2001,
pagine: 55 - 65
SICI:
1046-5928(200110)23:1<55:EACORH>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
ANTI-THROMBIN-III; HIGH-AFFINITY HEPARIN; METHYLOTROPHIC YEAST; HUMAN-PLASMA; SACCHAROMYCES-CEREVISIAE; PLASMINOGEN-ACTIVATOR; SECRETORY PRODUCTION; 384 ALA; VARIANT; BINDING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
44
Recensione:
Indirizzi per estratti:
Indirizzo: Mochizuki, S Welfide Corp, Div Pharmaceut Res, Drug Discovery Labs, 2-25-1Shodaiohtani, Osaka 5731153, Japan Welfide Corp 2-25-1 Shodaiohtani OsakaJapan 5731153 , Japan
Citazione:
S. Mochizuki et al., "Expression and characterization of recombinant human antithrombin III in Pichia pastoris", PROT EX PUR, 23(1), 2001, pp. 55-65

Abstract

Antithrombin III (ATIII) is a member of the serpin superfamily and a majorregulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight bySDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop wasassumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 22/01/20 alle ore 06:58:57