engleski

Surface display of heterologous proteins in the yeast cell wall and their application in biotechnology

In this work three genetic cassettes consisting of a host promoter (GAL1 or PHO5), signal sequence for directing the protein into the secretory pathway, cell wall anchoring sequence for N-terminal or C-terminal immobilisation of the recombinant protein and genetic tags which enable detection of the recombinant protein were constructed. These cassettes allow easy insertion of any gene of interest by restriction cloning method and concomitant surface display of recombinant protein in yeast. The S. cerevisiae gene GRE3 coding for xylose reductase (XR) and Pseudomonas sp. ADP genes coding for atrazine degrading enzymes atzA, atzB, atzC and atzD were inserted in the prepared genetic cassettes. Western blot analysis confirmed that all recombinant XRs and three out of six recombinant atrazine degrading enzymes were successfully immobilized at the cell surface. Both immobilisation approaches were successful in localization of active XR at the cell surface. Either N- or C-terminal immobilisation did not change the substrate preference of the enzyme, but resulted in decreased affinity of recombinant XRs for both substrate and coenzyme NADPH. Recombinant XRs had the same pH-optimum around pH 5 and temperature-optimum around 60 0C. Furthermore, all recombinant XRs were not particularly sensitive to the addition of EDTA or non-ionic detergents but they were sensitive to the addition of PMSF, -mercaptoethanol, SDS and ethanol. Pir-XR immobilised recombinant enzyme showed higher stability than XR-GPI upon all conditions tested. In the other part of this work was shown that the cell wall capacity for binding heterologous proteins is limited by the amount of autochthonous cell wall proteins and that is possible to control precisely the expression level of the recombinant enzyme. However, the activity of surface displayed atrazine degrading enzymes was not detected probably due to miss-folding of the recombinant enzymes.

yeast cell wall ; cell wall proteins ; yeast surface display ; Ccw12 ; Pir4 ; xylose reductase ; enzymes for atrazine degradation

nije evidentirano