A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris
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This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Muts Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25 % of the total proteins in the culture supernatant, after 120 h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45 % of the total culture supernatant proteins) with concomitant reduction in the induction time to 48 h. The amount of BbST obtained was 148 mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Muts strains of P. pastoris.
KeywordsAOX-1 promoter α-Factor signal sequence Bubaline somatotropin Methanol induction Pichia pastoris Polysorbate-80
This work was supported by a Grant from Higher Education Commission, Government of Pakistan.
- 7.Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Fact 9:31CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Inan M, Chiruvolu V, Eskridge KM, Valasuk GP, Dickerson K, Brown S, Meagher MM (1999) Optimization of temperature-glycerol-pH conditions for a fed-batch fermentation process for recombinant hookwork (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris. Enzyme Microb Technol 24:438–445CrossRefGoogle Scholar