engleski

Modification of protein synthesis and secretory pathway to improve surface display efficiency

Major disadvantages of protein surface display in yeasts as an alter-native to classical solid- surface immobilization techniques are low production, secretion, and binding capacity, or simply said low surface display efficiency of recombinant proteins. As several problems could lead to low display efficiency, we attempted to investigate how modification of different cellular processes involved in the production and secretion of proteins can affect and possibly enhance display efficiency. For this purpose, we tested the effects of mutations of genes involved in transcription and translation (deltagcn2, deltagc- n5, deltaopi1, deltarrp6) and mutations affecting properties of the endoplasmic reticulum (deltayop1). We also tested the effect of mutations of genes involved in unfolded protein response (UPR) and the endoplasmic reticulum- associated degradation (ERAD) pathways that are possibly activated due to stress conditions caused by increased protein production of overexpressed recombinant protein.To test the effect of these mutations we used two reporter systems previously developed in our laboratory. Both systems contain beta-lactamase fused with either Pir2 protein or with the GPI anchoring signal sequence of Ccw12. Results obtained by measuring the activity of surface-displayed beta-lactamase have shown that it was possible to increase surface display efficiency significantly by modifying processes involved in protein production and secretion in yeast cells.

Yeast, Surface Display, ER, transcription/translation regulation, UPR, ERAD

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