A Strategy to Increase the Intracellular Overexpression of Recombinant Proteins by Expressing Pichia pastoris Translation-related Factors
Article
Figures
Metrics
Preview PDF
Reference
Related
Cited by
Materials
Abstract:
This study was to screen the translation-related factors that promote the expression of recombinant proteins in Pichia pastoris, and amplify gene fragments of five types of translation-related factors (a total of 28) in Pichia pastoris (including ribosomal proteins, translation initiation factors, elongation factors, aa-tRNA synthetases, ribosome biogenesis factors, and ribosome-associated chaperones). These gene fragments were cloned into the pPICZA vector through homologous recombination. The recombinant vectors were further obtained and transformed into the Pichia pastoris GS115 strain with enhanced green fluorescent protein (eGFP) as the reporter protein. Then, the expression levels of 28 strains with overexpressed translation-related factors within 120 h were measured and calculated, to identify the key factors that could potentially promote the expression of the recombinant proteins in Pichia pastoris. Finally, the red fluorescent protein, monomeric red fluorescent protein (mRFP), was used as the reporter protein for verification. The results revealed that 6 translation-related factors (eIF4A, eEF1A, eEF3, Ded81, Bcy1, Ssb) that could improve the intracellular expression of recombinant proteins in Pichia pastoris, were screened in this study. Among them, the eGFP unit cell expression of eIF4A, eEF1A, eEF3, Ded81, Bcy1, Ssb increased by 18.40%, 18.80%, 29.50%, 28.45% and 21.60%, respectively, and the mRFP unit cell expression increased by 20%, 8.00, 19.00%, 5.40% and 15.40%, respectively. Bcy1 increased the biomass of eGFP-expressing strains by 20.00% and the biomass of mRFP-expressing strains by 30.90%. These results provide ideas for improving the expression of recombinant proteins in Pichia pastoris through the translation.
