Covalent immobilization and solid-phase refolding of enterokinase for fusion protein cleavage

Suh, Chang Woo; Park, Sin Hye; Park, Seung Gook; Lee, Eun Kyu

doi:10.1016/j.procbio.2004.06.050

Cited by 15 publications

(5 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Surprisingly, it was found that the undesirable side reaction, nonspecific cleavage especially, was significantly reduced. However, the activity of the N-terminal immobilized EK was only 20-30% of that of the free EK at various pH conditions (Suh et al 2005). A possible reason is that the N-terminal domain of EK plays an important role in the cleavage reaction (Choi et al 2001;Yuan and Hua 2002).…”

Section: Resultsmentioning

confidence: 97%

See 1 more Smart Citation

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in Escherichia coli

Gao

et al. 2010

World J Microbiol Biotechnol

View full text Add to dashboard Cite

As a promising type 2 anti-diabetic agent, glucagon-like peptide-1 (GLP-1) is attracting more and more interest. Mutated GLP-1 (mGLP-1) is an analog of native GLP-1. To facilitate the production and purification of mGLP-1, auto-induction and on-column cleavage was employed in this study. By using auto-induction system, after 24 h of shaking culture, about 12.6 g wet bacterial cells could be obtained from 1 l medium, and this was about 3.6 times more than that of the IPTG-induction group. After disruption and centrifugation, the fusion protein was directly purified and cleaved on Ni-Sepharose 6 Fast Flow column. Then, RESOURCE15 RPC column was used for further purification. By using these two steps of purification, about 1.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 g wet bacterial cells. In the bioactivity study, mGLP-1 displayed a significant and dose-dependent glucose-lowering activity. These results suggested that auto-induction and on-column cleavage could facilitate the production and purification of mGLP-1. These methods could also be applied to the preparation of other proteins and peptides.

show abstract

Section: Resultsmentioning

confidence: 97%

“…The undesirable cleavage was found to be significantly reduced when EK was covalently immobilized to glyoxyl agarose. However, the specific activity of immobilized EK was only 20-30% of that of the free enzyme (Suh et al 2005). How to improve specificity of EK while maintaining the specific activity of this enzyme remains unsolved.…”

Section: Introductionmentioning

confidence: 99%

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in Escherichia coli

Gao

et al. 2010

World J Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…As shown in Figure , both immobilized preparations showed no reduction in catalytic performance over a period of 20 days. It is worth noting that enzymatic preparation with the single-point attachment of NK14-AP per molecule ensures an operational stability of chemical preparation comparable with multipoint attachment to the supports . The results provide further evidence of the covalent bond formation of NK14-AP to the casein-grafted agarose beads and clearly show the potential of MTG-mediated enzyme immobilization.…”

Section: Resultsmentioning

confidence: 59%

Design of a Specific Peptide Tag that Affords Covalent and Site-Specific Enzyme Immobilization Catalyzed by Microbial Transglutaminase

et al. 2005

View full text Add to dashboard Cite

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

show abstract

“…Also, thermolysin has been immobilized with such purpose and a solution containing sodium and potassium acetate was selected as refolding medium obtaining a 100% yield of recovery of enzyme activity (Nohara et al, 2000). Similar results in terms of yield were obtained with immobilized chymotrypsin and trypsin partly inactivated by incubation at high temperatures (Guisán et al, 1992;Klibanov and Mozhaev, 1978;Martinek et al, 1980a,b;Mozhaev and Martinek, 1982;Mozhaev et al, 1987;Soler et al, 1997), with lipases (Guisán et al, 1996) and also with a recombinant enterokinase (Suh et al, 2005). In all these cases immobilization favored refolding and the most adequate medium for refolding was determined.…”

Section: Introductionmentioning

confidence: 72%

Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Romero

Vergara

Fernández‐Lafuente

et al. 2009

Biotech & Bioengineering

View full text Add to dashboard Cite

Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross-linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re-incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation.

show abstract

Covalent immobilization and solid-phase refolding of enterokinase for fusion protein cleavage

Cited by 15 publications

References 18 publications

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in Escherichia coli

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in Escherichia coli

Design of a Specific Peptide Tag that Affords Covalent and Site-Specific Enzyme Immobilization Catalyzed by Microbial Transglutaminase

Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Contact Info

Product

Resources

About