A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris.
This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut(s) 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 Mut(s) strains of P. pastoris.
Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54590, Pakistan, sasadaf@hotmail.com.
Saima
S
Sadaf
Hammad
H
Arshad
M
M
Waheed Akhtar
D042931:Aldehyde Oxidase / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D011136:Polysorbates / Q000008:administration & dosage; D051381:Rats; D013501:Surface-Active Agents / Q000008:administration & dosage*
D003470:Culture Media; D011136:Polysorbates; D013501:Surface-Active Agents; D013006:Growth Hormone; D042931:Aldehyde Oxidase