Expression And DNA Shuffling Of Recombinant Human Antithrombin â…¢ (AT-â…¢) In Pichia Pastoris

AT-â…¢ is an important plasma glycoprotein responsible for thrombin inhibition in blood coagulation cascade, and AT-â…¢ pharmaceutial products purified from plasma have been greatly used in clinic. Because of the high risk of spread of infectious virus, the plasma-derived AT-III is to be replaced by the recombinant AT-â…¢.The aim of this paper was to study the expression of human recombinant AT-III in Pichia pastoris-based system. The experiment was composed of four parts:(1) Construction of vector pPIC9K-ATHI The primers were designed with computer program. There were two kinds of primers leading to vector ' s signal and AT-III native signal respectively. Human AT-III gene was amplified from the constructed vector pcDNAS. 1-ATâ…¢ and confirmed by automatic DNA sequencing. Then AT-III cDNA was inserted into the expression vector pPIC9K, and the plasmid pPIC9K-AT was constructed. (2) Transformation and expression The Pichia pastoris GS115 strain was transformed with linearized plasmid pPIC9K-AT through electroporation. AT-III expressed in culture supernatant was confirmed by functional biological assay, western bloting and SDS-PAGE. Then culture condition of the strain was optimized to increase the yield or decrease the cost. (3) Purification The culture medium was centrifuged, and the supernatant was filtered with a 0. 22Hm filter and applied to the Heparin-hyperD column. The purity of AT-III was tested to be higher than 95% by SDS-PAGE. (4) DNA shuffling of AT-III The gene encoding AT-â…¢ was partially digested withDNase I . A single product with the correct size is amplified by self-priming PCR and reassembly PCR. Then a library of AT-â…¢ gene shuffled was constructed. The gene shuffled was transformed into Pichia pastoris and expressed and 15 high biological activity colonies have been screened and so far. So, AT-â…¢ was expressed in Pichia pastoris succesfully, and primary study of DNA shuffling has been done.