Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography.
During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.
Lienqueo
M E
ME
Centre for Biochemical Engineering and Biotechnology, Institute for Cell Dynamics and Biotechnology, Department of Chemical Engineering and Biotechnology, University of Chile, Beauchef 861, Santiago, Chile. mlienque@ing.uchile.cl
Salazar
O
O
Calado
C R C
CR
Fonseca
L P
LP
Cabral
J M S
JM
D000327:Adsorption; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D014364:Tryptophan / Q000378:metabolism*
D011994:Recombinant Proteins; D014364:Tryptophan; D002265:Carboxylic Ester Hydrolases; C019952:cutinase