YEAST SURFACE DISPLAY OF HUMAN PROTEASOME SUBUNIT ALPHA 6 AND EXPRESSION ENHANCEMENT

To construct the yeast display system of the human proteasome subunit alpha 6 (hPSA6) for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway, and enhance the display expression of hPSA6, the gene PSA6_HUMAN coding hPSA6 was cloned into a yeast displaying expression vector, pICAS H, which had been inserted a His, tag marker for expression level detection. As probed with a his, tag monoclonal antibody (MAb) and corresponding MAb, hPSA6 was detected functionally by flow cytometry and fluorescence microscopy analysis which confirmed that yeast-displaying recombinant hPSA6 with highly specific affinity was expressed efficiently after 24 h cultivation of recombinant yeast MT8-1/pICAS-H-PSA6. Induced by different concentration of initial glucose and case in acid in restrictive mediums, the displayed hPSA6 expressions were compared through immunofluorescence by using anti-His MAbs. Comparing with negative control, more than 70% cells were induced to express hPSA6 in SA with 3% casein acid initially. The 3% glucose could make more than half cells express well and nearly 40% cells were displaying hPSA6 successfully under the 2% initial glucose. Considering the glucose effect and the carbonization in sterilization period, 2% glucose was more appropriate than 3% in hPSA6 displaying.