Expression and characterization of recombinant human antithrombin III in Pichia pastoris.
ABSTRACT Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator 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 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 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 was assumed 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.
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ABSTRACT: Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. Antithrombin (AT) is a member of the serine protease inhibitor superfamily and a major coagulation inhibitor in all vertebrates, but its evolutionary origin remains elusive. In this study we isolated for the first time a cDNA encoding an antithrombin homolog, BjATl, from the protochordate Branchiostoma japonicum. The deduced protein BjATl consisted of 338 amino acids sharing 36.7% to 41.1% identity to known vertebrate ATs. BjATl contains a potential N-linked glycosylation site, two potential heparin binding sites and the reactive center loop with the absolutely conserved sequence Gly-Arg-Ser; all of these are features characteristic of ATs. All three phylogenetic trees constructed using Neighbor-Joining, Maximum-Likelihood and Bayesian-Inference methods also placed BjATl together with ATs. Moreover, BjATl expressed in yeast cells was able to inhibit bovine thrombin activity by forming a SDS-stable BjATl-thrombin complex. It also displays a concentration-dependent inhibition of thrombin that is accelerated by heparin. Furthermore, BjATl was predominantly expressed in the hepatic caecum and hind-gut, agreeing with the expression pattern of AT in mammalian species. All these data clearly demonstrate that BjATl is an ortholog of vertebrate ATs, suggesting that a primitive coagulation system emerged in the protochordate.PLoS ONE 01/2012; 7(3):e32392. · 3.73 Impact Factor
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ABSTRACT: Expression of recombinant pharmaceutical proteins in the mammalian mammary gland is of great interest for the medical industry. This study was designed to express recombinant human antithrombin (rhAT) in the mammary gland of rabbits by adenovirus vectors infection. Replication-defective adenovirus encoding human antithrombin complementary DNA (cDNA) was constructed and directly infused into the mammary gland of rabbits via the teat canal. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to Western blot analysis, Enzyme-linked immunosorbent assay (ELISA), and antithrombotic activity assay. In this way, the target protein was verified, and a high expression level of rhAT up to 4.8 g/L was obtained, and antithrombotic activity of the rhAT was not different than that of a standard human antithrombin protein (p > 0.05). Compared to previous attempts to produce human antithrombin in the mammary gland of transgenic animals or fractionation the plasma of blood donors, the method for rhAT expression we established would reduce production cost and further increase production efficacy.Animal Biotechnology 04/2012; 23(2):89-100. · 0.88 Impact Factor
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ABSTRACT: Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development. However, detailed understanding of the yeast's physiology, genetics and regulation has only developed rapidly in the last few years since published genome sequences have become available. An expanding toolbox of genetic elements and strains for the improvement of protein production is being generated, including promoters, gene copy-number enhancement, gene knockout and high-throughput methods. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. At the same time, it has become obvious that P. pastoris, as an evolutionarily more 'ancient' yeast, may in some cases be a better model for human cell biology and disease than Saccharomyces cerevisiae.Future Microbiology 02/2013; 8:191-208. · 4.02 Impact Factor