Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography

Abstract

Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

“…The surface hydrophobicity of tagged proteins (φ tagged ) has been used by Lienqueo et al (2007) for predicting the DRT of cutinases tagged with hydrophobic peptides in different matrices for HIC, by means of equation (13) and the methodology represented in Figure 3. The coefficients of the linear model are constants for each set of operating conditions.…”

“…The simplest methodology uses straightforward quadratic models, whose parameters depend on the chromatographic conditions used in the HIC run (Lienqueo et al, 2007), and whose variables are DRT and the average surface hydrophobicity of the protein to be separated (φ surface ). The most appropriate hydrophobicity scale was found to be that proposed by Miyazawa & Jernigan (1996), in its normalized form.…”

“…The suitability of the hydrophobicity scale depends on the use that will be given to the estimation of the protein or peptide hydrophobicity, as well as on the way to estimate this property. The scales proposed by Miyazawa & Jernigan (1996) and by Cowan & Whittaker (1990) are the most adequate to estimate protein hydrophobicity based on its threedimensional structure (Lienqueo et al, 2007), regarding its behavior in HIC. Additionally, Salgado et al (2005) proposed that the scale developed by Wertz & Scheraga (1978) is the most adequate to estimate protein hydrophobicity based on the amino acid composition of that protein.…”

“…Glycine usually has an intermediate hydrophobicity level, i.e. neutral hydrophobicity, and the lowest level is mostly assigned to Aspartic acid (Lienqueo et al, 2007), i.e., this is the most hydrophilic amino acid. The suitability of the hydrophobicity scale depends on the use that will be given to the estimation of the protein or peptide hydrophobicity, as well as on the way to estimate this property.…”

“…Table 2 shows the average surface hydrophobicity for a group of proteins using the amino acid hydrophobicity scales given in Table 1, and calculated by equation (10). This method for estimating protein hydrophobicity has proven to be valid in several cases (Lienqueo et al, 2002;Lienqueo et al, 2003;Lienqueo et al, 2007); however, this methodology is not valid for proteins that exhibit a highly heterogeneous distribution of the hydrophobic patches on their surfaces (Mahn et al, 2004 Table 2. Surface hydrophobicity of proteins estimated by equation (9).…”