Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase

Yeon, Young Joo; Park, Hyun June; Park, Hyung-Yeon; Yoo, Young Je

doi:10.1007/s12257-014-0089-2

Cited by 17 publications

(17 citation statements)

References 16 publications

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“…The His tag, S Tag and thrombin site at the N‐terminal of the recombinant exo‐inulinase may influence the post translation modification and possibly increase the catalytic efficiency by allowing for easier access of inulin and sucrose to the active site of recombinant enzyme. Yeon et al found the recombinant 3‐hydroxybutyrate dehydrogenase enzyme to be 1,200‐fold more active with N‐terminal His‐tag when compared to C‐terminal His tag. Also, glycosylation may play a role in the post translation modification of the protein.…”

Section: Resultsmentioning

confidence: 99%

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Yedahalli

Rehmann

Bassi

2016

Biotechnology Progress

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Inulin is a linear carbohydrate polymer of fructose subunits (2-60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo-oligosaccharides, biofuel, and nutraceuticals. Cell-free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo-inulinase was investigated in this study. The eukaroyototic exo-inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta-gami B (DE3)]. The molecular weight of recombinant exo-inulinase was estimated to be ∼81 kDa. The values of Km and Vmax of the recombinant exo-inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min(-1) mg(-1) protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min(-1) mg(-1) protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo-inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo-inulinase activity towards inulin was enhanced by Cu(2+) and reduced by Fe(2+) , while its activity towards sucrose was enhanced by Co(2+) and reduced by Zn(2+) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629-637, 2016.

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Section: Resultsmentioning

confidence: 99%

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Yedahalli

Rehmann

Bassi

2016

Biotechnology Progress

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“…In this method, the purified SpA fraction derived from high water/low acetonitrile elution solvent is the correctly folded form of SpA, which retains the desired bioactivity. Additionally, a His-tag might affect the function of the recombinant protein and when the recombinant protein is to be used as drug the tag should be removed [15]. The purification of SpA was also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis.…”

Section: Production Of Protein Amentioning

confidence: 87%

“…[14][15][16][39][40][41][42][43][44] with MabSelect [31]. Some cellular proteins contain two or more adjacent histidine residues which may interfere with affinity purification.…”

Section: Per Aqueous Liquid Chromatography (Palc)mentioning

confidence: 99%

A comprehensive review on staphylococcal protein A (SpA): Its production and applications

Rigi

Ghaedmohammadi

Ahmadian

2019

Biotech and App Biochem

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The Staphylococcus aureus protein A (SpA) can be obtained through the culture of wild-type S. aureus and also as a recombinant protein in safe bacterial hosts. Several methods have been used to purify SpA among which ion-exchange chromatography, affinity chromatography, gel filtration, and per aqueous liquid chromatography (PALC) are common. SpA has a wide range of biochemical, biotechnological, and medical applications and is most commonly used in test methods such as immunoprecipitation, enzyme-linked immunosorbent assay, and Western blotting. SpA has also been widely utilized in pharmaceutical applications to bind to immune complexes and serum immunoglobulins. SpA also directly binds to the B-cells preventing initiation of infectious diseases as well as having a role in the development of various autoimmune diseases. This review considers different applications of SpA in biotechnology and its novel clinical application for effective treatment of autoimmune diseases. It also discusses various strategies for expression and purification of the SpA including types of column chromatography that are commonly used in protein purification and developing SpA surface display technologies. Finally, this review highlights the potential and novel applications of SpA immobilization, SpA typing, protein engineering for further development of immunological and biochemical research, and also application of SpA as a diagnostic biosensor.

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“…The PCR product with a stop codon was digested with Nde I and Hind III, and then was ligated into similarly digested pET22b (without signal peptide). The resultant plasmid was transformed into E. coli BL21 (DE3) for expression of wild‐type PcEST without a his‐tag to avoid the negative influence of the tag on enzymatic activity …”

Section: Methodsmentioning

confidence: 99%

“…The bound protein was eluted with elution buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 250 mM imidazole, 1 mM β‐mercaptoethanol). The 6×his‐smt3 tag of the protein was excised by ULP protease in dialysis buffer (10 mM Tris‐HCl pH 8.0, 100 mM NaCl, 1 mM β‐mercaptoethanol) at 4 °C to avoid the negative effect of tag on enzymatic activity . Finally, the proteins were loaded onto Ni–NTA column pre‐equilibrated with dialysis buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM β‐mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

et al. 2018

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Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 °C increased T over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale.

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Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase

Cited by 17 publications

References 16 publications

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

A comprehensive review on staphylococcal protein A (SpA): Its production and applications

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

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