Heterologous expression of extracellular proteins by Yarrowia lipolytica

[摘要] English: The C. fumago chloroperoxidase (CPO) and human Granulocyte-Colony Stimulating Factor (hG-CSF) proteins require post-translation glycosylation modifications, contain intra molecular disulfide bonds for proper folding and biological activity, and have presented challenges when heterologously expressed in yeast, bacterial, insect cells and mammalian expression systems. The constraints associated with recombinant production of the proteins included toxicity to the host cell, inappropriate post-translational modifications, low production yields, improper protein folding, and lack of biological activity. The objective of this study was to explore the versatility of Y. lipolytica as host for expression and extracellular secretion of these two commercially significant proteins. The Y. lipolytica yeast was selected for its inherent ability to secrete a variety of proteins, its reported cotranslational translocation protein secretion pathway similar to that of mammalian systems, low overglycosylation, high protein secretion efficiency, good protein product yield, and heterologous protein production performance reproducibility. The codon optimized synthetic CPO and hG-CSF genes were cloned under the quasi constitutive growth dependent HP4D promoter or the inducible POX2 promoter in multicopy zeta based integrative expression systems using the Trimetes vesicolor laccase (LACC) or Y. lipolytica extracellular lipase (LIP2) secretion signals to target protein expression to the extracellular.The CPO protein was expressed as an inactive extracellularly located protein judging by Western blot analysis using poly-Histidine antibody. The antibody detected the His tag fused to the C-terminus of the recombinant protein. The detection of this His tag suggested the failure by the Y. lipolytica to process through proteolytic cleavage, the 52 amino acid propeptide located at the C-terminal end of the protein. The 52 amino acid propeptide acts like a chaperone, being required to ensure correct CPO protein maturation, and its removal at a later stage during CPO secretion is required to yield an active CPO enzyme. The recombinant hG-CSF protein was successfully expressed in Y. lipolytica and purified by Ni-NTA affinity chromatography to homogeneity as judged by the single protein band with relative molecular mass of about 18 kDa. The protein was confirmed to be hG-CSF by peptide mapping using LC-MS/MS analysis. The biological activity of the expressed hG-CSF was found to be very low in comparison with the commercially available nonglycosylated hG-CSF. It remains to be determined if the His tag fused to the C-terminal end of the recombinant protein affected the biological activity of the recombinant hG-CSF. The possible explanation for the low biological activity could be the cultivation conditions that did not take into account pH ranges at which hG-CSF retains stability.The study demonstrated the potential application of the Y. lipolytica yeast as platform for production of recombinant CPO and hG-CSF proteins. The proteins were successfully expressed and secreted to the extracellular. However, the SDS-PAGE analysis revealed that the CPO on the extracellular was less pronounced as compared to hG-CSF protein. Most importantly, this study provided insights on the requirements that have to be considered when developing the Y. lipolytica yeast as production systems for recombinant CPO and hG-CSF. The future attempts in heterologous expression of CPO enzyme in Y. lipolytica need to include development of platforms to co-express compatible endoprotease genes to enable efficient processing of the chaperonic 52 amino acid long carboxyl terminal propetide. The factors to be considered in future studies on the production of hG-CSF protein would entail the design of appropriate cultivation conditions to enhance the stability of hG-CSF during production, and the use of Y. lipolytica strains deleted for the yeast glycosylation pathway.

[发布机构] University of the Free State