Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeastPichia pastoris

Abstract

Aims: This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization. Methods and Results: Multi‐copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real‐time qPCR. A range of Mut+P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell‐shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l−1 was achieved by a 12‐copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (>12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress. Conclusions: A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (>12 copies) significantly impaired the growth of P. pastoris hosts. Significance and Impact of the Study: The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA inPichia pastoris:sequence modifications and a simple method for the generation of multi-copy strains

“…These results are comparable with those reported by Zhu et al (2009) [13], where production of a recombinant protein using a methanol-induced vector in P. pastoris increased approximately 10-fold when increasing gene copy number from 1 to 12, but decreased when vectors with higher copy numbers were introduced. In addition Zhu et al, [13] found a reduction in cell growth rate with strains carrying more than the optimal 12 copies of the transgene.…”

“…In addition Zhu et al, [13] found a reduction in cell growth rate with strains carrying more than the optimal 12 copies of the transgene. In this study a reduction in cell growth rate was noted for SMD1168H cells carrying more than 5 copies of the transgene but this was not observed for X33 cells.…”

“…Phusion polymerase was from New England BioLabs. GNA was produced as a recombinant protein in yeast using a clone generated as previously described ( [13] Raemaekers et al, 1999). Polyclonal anti-GNA antibodies (raised in rabbits) were prepared by Genosys Biotechnologies, Cambridge, UK.…”

“…To ensure multiple copies of recombinant gene carried by the vector are incorporated into the genome of P. pastoris, the approach followed was to produce vectors carrying multiple copies of the expression construct, using a strategy based on that described by Zhu et al, [13]. This approach is much more reliable than the earlier method of screening large numbers of transformants for "jackpot" high copy strains resulting from multiple integrations of a transforming plasmid ( [23] Higgins and Cregg, 1998).…”

“…al., 2001, Mansur et. al., 2005; with recombinant protein production increasing with increasing copy number to an optimum, after which further increasing the copy number results in decreased expression ( [13] Zhu et. al., 2009).…”