Expression and characterization of recombinant human antithrombin III in Pichia pastoris

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human AT III (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature p rotein region connected with the truncated mAOX2 promoter and the SUC2 secr etion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant re ached a level of 320 IU/L in the culture broth at 169 h. From the culture-s upernatant, rATIII was purified to over 99% by heparin-affinity chromatogra phy and other column chromatography methods. We characterized rATIII and co mpared it with human plasma-derived ATIII (pATIII). The purified rATIII pos sessed correct N-terminal amino acid sequence, and its molecular weight by SDS-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 pos ttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification w ith O-linked mannosyl-mannose had probably occurred at Thr 386, close to th e reactive center. Although the heparin-binding affinity of rATIII was 10-f old higher than that of pATIII, its inhibitory activity against thrombin wa s only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pAT III can inactivate thrombin through formation of a stable thrombin-ATIII co mplex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin. (C) 2001 Acad emic Press.