engleski

INCREASING THE EFFICIENCY OF PIR2-BASED YEAST SURFACE DISPLAY SYSTEM

BACKGROUND Saccharomyces cerevisiae is frequently used as a host for the production of recombinant proteins. S. cerevisiae also provides the immobilization of recombinant proteins on the cell surface as an alternative to conventional immobilization methods. Yeast surface display systems are based on genetic fusion of a gene coding for a protein of interest and a gene coding for one of yeast cell wall proteins. Since immobilization of the protein through its N- or C- terminal end can affect its activity, different surface display systems were developed. Proteins of the Pir family are standardly used for N- terminal immobilization of recombinant proteins. OBJECTIVES Pir-based display systems developed so far involve fusion of the whole Pir protein with the desired protein, resulting in a large recombinant protein whose expression cause problems in its secretion and cell wall binding. To address this problem, we introduced changes in the Pir2 sequence and the protein secretion mechanisms to increase the efficiency of the Pir2- based surface display system. METHODS Using bacterial β-lactamase as a reporter enzyme, we assessed the efficiency of surface display of non-modified and modified recombinant protein Pir2bla in different yeast mutants. Amount of protein incorporated in cell wall was assessed by measurement of β-lactamase activity and by western blot. RESULTS Results show higher β-lactamase activity in strains expressing recombinant proteins containing the modified Pir2 binding region, lacking multiple internal repeats, and an increase of activity in most of the tested strains lacking activity of certain genes involved in endoplasmic reticulum-associated degradation (ERAD) pathways, or endocytosis. ACKNOWLEDGEMENTS/REFERENCES This research was funded by The Croatian Science Foundation, grant No. IP-2019-04-2891.

recombinant proteins, surface display, Pir2

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