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Effects of mutations in endocytosis and glycosylation on efficiency of surface display of recombinant proteins in yeast

Surface display in yeast provides an alternative to standard protein immobilization on solid surfaces as it reduces the costs of protein purification and possibility of protein denaturation during the immobilization. Also, it provides continuous synthesis of recombinant protein composed of enzyme of interest fused with native yeast cell wall proteins. However, its major disadvantage is low surface display efficiency. Lately, it has been reported that mutations in genes encoding proteins involved in endocytosis may increase the amount of secreted heterologous proteins from the yeast cells. Glycosylation can also affect the efficiency of surface display as it could possibly have negative effect on conformation of displayed proteins and cause high density of wall outer mannan layer, limiting accessibility of the substrate. In order to test whether inactivation of genes involved in these processes would enhance surface display efficiency, two different systems using β-lactamase as a reporter were developed. In one, bla gene was fused with a fragment of CCW12 gene coding for its C-terminal GPI anchoring signal sequence. Other system consisted of bla gene coding for β-lactamase fused with PIR2 gene coding for cell wall protein that covalently binds to cell wall through the linkage on its N-terminal end. Both constructs were set under the control of an inducible PHO5 promotor. By using two different immobilization systems, the effect of different folding of the recombinant enzyme, caused by immobilization itself, on affecting enzyme activity is reduced. Therefore, the described system for expression and immobilization of recombinant reporter enzyme in the wall was examined in mutants that cannot accomplish endocytosis (end6, end3, vam4) and have lower level of glycosylation (pmt and mnn mutants). Amount of recombinant protein incorporated in cell wall was assessed by measurement of β-lactamase activity using nitrocefin as substrate and in a semi- quantitative manner by western blot.

Surface display, β-lactamase, Ccw12, Pir2, glycosylation, endocytosis

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