Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae
Artikel i vetenskaplig tidskrift, 2012

Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and a-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and a-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (mu mol/L) of IP was found to be higher than that of a-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.

secretory pathway

insulin precursor

secretory expression

directed evolution

endoplasmic-reticulum

gene-expression

a-amylase

heterologous proteins

Saccharomyces cerevisiae

binding-protein

leader sequence

yeast

expression systems

insulin precursor

heat-shock

ethanol stress

Författare

Zihe Liu

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Keith Tyo

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Jose Luis Martinez Ruiz

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Dina Petranovic Nielsen

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Jens B Nielsen

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

Biotechnology and Bioengineering

0006-3592 (ISSN) 1097-0290 (eISSN)

Vol. 109 1259-1268

Ämneskategorier

Industriell bioteknik

Mikrobiologi

Styrkeområden

Livsvetenskaper och teknik

DOI

10.1002/bit.24409