研究报告 |
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Inhibitor of Neutrophil Proteinases Elafin Expression in Pichia pastoris and Activity Investigation |
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Abstract Elafin is a specific inhibitor of neutrophil elastase.To establish expression system of neutrophil proteinases inhibitor elafin in Pichia pastoris , Elafin cDNA was obtained by reverse transcription PCR(RT-PCR).With restricted enzyme digestion,gene of interest was cloned into shuttle vector pPIC9K.Then the construct was linearized and transformed into Pichia pastoris competent cells by electroporation.Selected positive clone was inoculated into buffered minimal glycerol(BMG)culture media.Add 100% methanol to a final concentration of 1.0% every 24 hours to maintain induction.At several different time points, transfer 1ml of expression cultrure to centrifuge to analyze the expression level by Coomassie-stained SDS-PAGE.Concentrated supernatnat was loaded onto a S6 column using a chromatographic system to purify secreted reconbinant elafin(rec-elafin).Rec-elafin immunogen and activity against proteinases was investigated by western blot and elastin-PAGE respectively. The rec-elafin expression level was up to 12 mg/L.Secreted elafin in Pichia pastoris was easily purified by one-step cationic exchange chromatography and retained its activity.
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Received: 26 June 2006
Published: 25 December 2006
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