Naslov
Surface display of recombinant xylose reductase in the Saccharomyces cerevisiae

Autori
Hossain, Sk Amir ; Teparić, Renata ; Mrša, Vladimir

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Power of Microbes in Industry and Environment 2016 / Mrša, Vladimir ; Teparić, Renata ; Kifer, Domagoj - Zagreb : Hrvatsko mikrobiološko društvo, 2016, 54-54

ISBN
978-953-7778-14-9

Skup
Power of Microbes in Industry and Environment 2016

Mjesto i datum
Krk, Hrvatska, 28.09.2016. - 01.10.2016

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
surface display; recombinant proteins; Saccharomyces cerevisiae

Sažetak
Most of the vectors constructed so far for the surface display of recombinant proteins in the yeast have been created with ubiquitous plasmids. We have constructed three new plasmids for the surface display of heterologous proteins in the yeast S. cerevisiae that would enable easy insertion of the gene of interest into a prearranged genetic cassette consisting of a strong and regulated host promoter, signal sequence for directing the protein into the secretory pathway, 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 the heterologous proteins, containing PIR4 under a GAL1 promoter followed by the spacer region (a stretch of eight serine residues), region consisting of several restriction sites and finally by the -6xHis and -HA tags. Plasmids pRS425Ccw12 and pRS425Ccw12_2 were prepared for C-terminal immobilisation of heterologous proteins. The pRS425Ccw12 plasmid contains GAL1 promoter 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 C-terminal part of Ccw12 containing the GPI anchoring signal and the downstream genetic elements of the CCW12. In the pRS425Ccw12_2 plasmid the -HA tag is placed after the heterologous gene insertion site, followed by the GPI anchoring signal and the downstream genetic elements of the CCW12. The gene GRE3 coding for xylose reductase was inserted into the plasmids described above using suitable restriction sites. Finally, the localization, abundance and the activity of the surface displayed xylose reductase were checked by Western blot method and spectrophotometric assay and the results obtained were compared to find out which type of immobilization is best for the surface display of this particular enzyme.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

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