Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography

Abstract

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.

Immobilization of Proteins in Poly-Styrene-Divinylbenzene Matrices: Functional Properties and Applications

“…A likely application of these hydrophobic supports, still to be reported is the one-step immobilization-purification of chimeric proteins bearing hydrophobic tags, some papers show how using hydrophobic supports, these tagged enzymes may be selectively immobilized [111][112][113]. These proteins should have a large affinity for these hydrophobic supports.…”

Challenges and opportunities in the purification of recombinant tagged proteins

“…Examples of hydrophobic tags for recombinant protein purification include tags based on tryptophan and tyrosine, exploited for aqueous two phase separation (ATPS) , and tags based on repetitions of the dipeptide tryptophan-proline (WP) n (Lienqueo et al, 2008). It has been reported that as the length of the tag increases, a negative effect on cellular growth, stability of the plasmids, secretion efficiency of the target protein and a decrease of recovery yield on purification stage are observed (Lienqueo et al, 2008).…”

“…Examples of hydrophobic tags for recombinant protein purification include tags based on tryptophan and tyrosine, exploited for aqueous two phase separation (ATPS) , and tags based on repetitions of the dipeptide tryptophan-proline (WP) n (Lienqueo et al, 2008). It has been reported that as the length of the tag increases, a negative effect on cellular growth, stability of the plasmids, secretion efficiency of the target protein and a decrease of recovery yield on purification stage are observed (Lienqueo et al, 2008). According to Collén et al, during protein expression, fully exposed hydrophobic tags can reduce target protein solubility, compromising the secretion pathway and leading to the formation of insoluble protein aggregates (Collén et al, 2001) and the chance that the target protein will be membraneassociated increases .…”