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Characetrisation of surface displayed recombinant xylose reductase in Saccharomyces cerevisiae

Two types of genetic cassettes for the surface display of heterologous proteins in S. cerevisiae were constructed and their functionality was tested by cloning recombinant enzyme xylose reductase (XR). Cassettes contained strong and regulated host promoter GAL1 or PHO5, a signal sequence for directing the protein into the secretory pathway, an anchoring domain of native cell wall proteins for C- or N-terminal immobilisation, and genetic tags for easy detection of the recombinant protein. YEp351Pir4 plasmid was constructed for the N-terminal immobilisation of heterologous proteins, containing PIR4 under a GAL1 promoter followed by the spacer region (a stretch of eight serine residues), a region consisting of several restriction sites for the insertion of the gene of interest and, finally, followed by the -6xHis and -HA tags. The plasmid pRS425Ccw12G and pRS425Ccw12P were prepared for C-terminal immobilisation of heterologous proteins. This plasmid contains a GAL1 or PHO5 promoter, respectively, followed by the part of CCW12 coding for the signal sequence, the -HA tag, restriction sites for the insertion of the gene of interest, the part of the CCW12 coding for the GPI anchoring signal, and the downstream genetic elements of the CCW12. The S. cerevisiae gene GRE3 coding for intracellular xylose reductase was inserted into the plasmids described above using suitable restriction sites. The construct pRS425Ccw12GXR was modified by introducing the STOP codon immediately after GRE3 coding region, resulting in a secretion of recombinant xylose reductase into the growth medium. Finally, the localization, activity and characteristics of different forms of recombinant xylose reductase were determined. Both types of cassettes proved to be functional, and for this recombinant protein N-terminal immobilisation showed to be better solution for surface display.

Ccw12, Pir4, Recombinant protein, Saccharomyces cerevisiae, xylose reductase

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