engleski

Altering the cell wall to increase cell surface display efficiency

Yeast surface display is a powerful and robust method for the surface immobilization of heterologous proteins with potential application in different biomedical and biotechnological processes. In biotechnology, it could be used in various bioconversion processes as enzymes of interest can be fused with anchoring proteins. This allows secretion of such protein constructs and their binding to the cell wall. The major disadvantage of this method is the low binding capacity and low overall surface dis-play efficiency of currently used systems. There are several possible approaches to resolve this problem, and in this work, we focused on the modification of the cell wall of host cells. Several mutant strains were developed and/or tested to see if it is possible to increase display efficiency by introducing changes in cell wall structures. Tested strains contained mutations in genes coding for cell wall proteins (the strain with deleted genes coding for Pir proteins, and deltacwp2 strain) or in genes coding for proteins involved in the synthesis of the GPI anchor, and in the localization of GPI anchored wall proteins (deltagpi7, deltadfg5, deltadcw1 strains). Also, the effect of deletion of the SSD1 gene coding for the translation factor involved in the translation of many cell wall proteins was tested. Effects of these mutations were investigated using reporter systems previously developed in our laboratory, composed of beta- lactamase fused with either Pir2 or Ccw12. Our preliminary results show that it is possible to obtain more than two-fold higher surface display efficiency by these modifications.

Yeast, Surface Display, GPI anchor, SSD1, Pir proteins

nije evidentirano