Engineering a single chain minimal HLA-DR4 by directed evolution: A quality control based approach to yeast surface display.

摘要

Yeast surface display of proteins has proven to be a useful protein engineering tool. Several proteins have been successfully surface-displayed and engineered for both improved stability and function through combinatorial libraries and directed evolution. Successful surface display has been correlated with enhanced stability and expression; however, no protein to date has been engineered for enhanced stability based solely on higher expression. Closer examination of a protein engineered for enhanced function, a single chain fragment (scFv) of the 4-4-20 antibody, has determined that enhanced function does not necessarily lead to enhanced stability. However, the wild type scFv expressed well and retained its affinity for ligand. In fact, the surface-displayed protein was heterogeneously truncated, and inhibition of the yeast proteasome alleviated the truncations, which marks the first notice of reversible retrotranslocation after targeted degradation.;Saccharomyces cerevisiae shares significant homology with mammalian cells, which possess strict quality control machinery to regulate the expression of proteins only capable of folding into native structures. A noncovalent class II major histocompatibility (MHC) heterodimer complexed with antigenic peptide has been displayed on the surface of yeast in a fully native form and represents the first example of a noncovalent protein dimer expressed by yeast surface display. In this thesis, a single chain minimal MHC was also examined; however, the wild type showed poor expression. Therefore, the truncated protein was engineered by directed evolution for mutants showing enhanced expression with the hypothesis that enhanced expression should lead to native-like protein folding.;Mutants with higher expression were isolated, but the set of mutations required for improved expression did not represent one, which would have anticipated based on the crystal structure of the full-length MHC. Data suggests that the protein is natively folded; however, attempts to solubly secrete the protein failed. A new method was developed to liberate the protein from the surface of the yeast cells. The processing required led to protein aggregation. As a result the structure of the protein has not been fully characterized. The difficulties encountered to produce soluble protein, which has been successfully surface-displayed, question the nature of yeast protein quality control.