Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

Kepka, Cecilia; Collet, Éric; Roos, Frederik C.; Tjerneld, Folke; Veide, Andres

doi:10.1016/j.chroma.2005.03.054

Cited by 27 publications

(12 citation statements)

References 22 publications

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“…Proteins may change conformation in the adsorbed state under strongly retaining conditions, as indicated by multiple or misshapen chromatographic peaks (Goheen and Engelhorn, 1984;Hahn et al, 2003;Jungbauer et al, 2005;Machold et al, 2002;To et al, 2007;Wu et al, 1986a,b), and increased solvent accessibility (Buijs et al, 1999;Fogle et al, 2006;Tibbs-Jones and Fernandez, 2003;Xiao et al, 2007b). This can reduce desired product recovery (Chen et al, 2007;Kepka et al, 2005;To et al, 2007), inhibiting the application of this purification method.…”

Section: Introductionmentioning

confidence: 92%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

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A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.

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Section: Introductionmentioning

confidence: 92%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

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“…The use of different molar ratios of phosphate salts of potassium and sodium do not affect binodal curve position. In addition, the high solubility of this system allows its use in salt stock solutions of elevated concentrations, required for industrial purpose [23]. Figure 1 shows the phase diagrams based on polyethylene glycol (PEG)/phosphate system used to calculate the tie line lengths (TLL) values aiming to evaluate G6PD purification in ATPS, which PEG concentrations varied in a range of 16 to 35 % (w/v) and phosphate concentration was kept constant at 12 % (w/v).…”

Section: Resultsmentioning

confidence: 99%

Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems

Gurpilhares

Pessoa

Roberto

2015

Appl Biochem Biotechnol

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Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells.

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“…There are some studies about the effect of the production and recovery of fusion cutinases (Calado et al 2002a;Cunha et al 2003;Kepka et al 2005;Sagt et al 1998). In order to obtain an efficient and low cost production system for cutinase from Fusarium solani pisi (wild-type cutinase), this enzyme can be overproduced in Saccharomyces cerevisiae.…”

Section: Introductionmentioning

confidence: 96%

“…Some of the most commonly used are tryptophan-containing tags (e.g. (WP) 2 , (WP) 4 ) (Bandmann et al 2000;Berggren et al 1999;Fexby and Bulow 2004;Kepka et al 2005;Rodenbrock et al 2000). Unfortunately, these short hydrophobic peptide tags could have some negative effects on the protein production and/or purification, for example: (a) hydrophobic fusion can cause the protein to be membrane-associated (Persson et al 1988); (b) proteolytic cleavage can reduce the amount of protein obtained (Collen et al 2001;Hassinen et al 1994); (c), changes in mRNA stability that will affect the expression levels (Fexby and Bulow 2004); (d) increased hydrophobic properties might result in protein association of dimers or larger aggregates (the tagged protein is partly/fully insoluble) (Sagt et al 1998), or in dissociation into smaller subunits (Johansson and Walter 2000); (e) the conformational changes might lead to denaturation and collapse of the protein stability loss (Terpe 2003).…”

Section: Introductionmentioning

confidence: 99%

“…The addition of hydrophobic tags is efficient in hydrophobic interaction chromatography (HIC) where the proteins can be eluted with different retention times (Hassinen et al 1994;Kepka et al 2005). This is a more cost-efficient chromatographic method for protein purification since HIC is less expensive compared to affinity chromatography based on ligands such IMAC, protein A and IgG.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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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.

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Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

Cited by 27 publications

References 22 publications

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems

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

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