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Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae

Zihe Liu (Institutionen för kemi- och bioteknik, Systembiologi) ; Keith Tyo (Institutionen för kemi- och bioteknik, Systembiologi) ; José Luis Martinez Ruiz (Institutionen för kemi- och bioteknik, Systembiologi) ; Dina Petranovic (Institutionen för kemi- och bioteknik, Systembiologi) ; Jens B. Nielsen (Institutionen för kemi- och bioteknik, Systembiologi)
Biotechnology and Bioengineering (0006-3592). Vol. 109 (2012), 5, p. 1259-1268.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: a-amylase, insulin precursor, expression systems, leader sequence, secretory pathway, Saccharomyces cerevisiae, heterologous proteins, secretory expression, gene-expression, endoplasmic-reticulum, directed evolution, insulin precursor, binding-protein, ethanol stress, heat-shock, yeast



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Denna post skapades 2012-04-26. Senast ändrad 2014-10-27.
CPL Pubid: 157111

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Systembiologi (2008-2014)

Ämnesområden

Livsvetenskaper
Mikrobiologi
Industriell bioteknik

Chalmers infrastruktur

Relaterade publikationer

Denna publikation ingår i:


Metabolic Engineering of Recombinant Protein Production by Saccharomyces cerevisiae


 


Projekt

Denna publikation är ett resultat av följande projekt:


Industrial Systems Biology of Yeast and A. oryzae (INSYSBIO) (EC/FP7/247013)