His-tag impact on structure

Carson, M.; Johnson, David H.; McDonald, Heather M.; Brouillette, Christie G.; DeLucas, Lawrence J.

doi:10.1107/s0907444906052024

Cited by 197 publications

(167 citation statements)

References 18 publications

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“…The GH43B6, which fused with histidyl tag at the C-terminus, retained the enzymatic function indicated that histidyl tag did not interfere the function of GH43B6. The presence of the histidyl tag had no significant effect on protein structure (Carson et al 2007). Thus, biochemical characterization of GH43B6 could be conducted without cleaving of histidyl tag.…”

Section: Saccharoperbutylacetonicummentioning

confidence: 99%

Multifunctional Properties of Glycoside Hydrolase Family 43 from Paenibacillus curdlanolyticus Strain B-6 Including Exo-β-xylosidase, Endo-xylanase, and α-L-Arabinofuranosidase Activities

Wongratpanya¹,

Imjongjairak²,

Waeonukul³

et al. 2015

BioResources

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Section: Saccharoperbutylacetonicummentioning

confidence: 99%

Multifunctional Properties of Glycoside Hydrolase Family 43 from Paenibacillus curdlanolyticus Strain B-6 Including Exo-β-xylosidase, Endo-xylanase, and α-L-Arabinofuranosidase Activities

Wongratpanya¹,

Imjongjairak²,

Waeonukul³

et al. 2015

BioResources

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“…However, no enzyme activity was detected when purified with NTA affinity column. This may be because His-tags generally have some minor effect on the structure of the native enzyme (Carson et al 2007). His-tags are known to mediate oligomerization via metal cation and can interact with some metal ions (Ca 2+, Mg 2+ ), which may affect the enzyme activity.…”

Section: Expression and Purification Of The Recombinant Enzymementioning

confidence: 99%

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Liu

Guo

et al. 2018

Bioresour. Bioprocess.

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Background: Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.Results: An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0-7.0 at 55 °C for 60 min. The enzyme exhibited K m , V max , K cat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 10 3 /S, respectively. The addition of 10 mM of Mg 2+ , Mn 2+ , or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity. Conclusions:An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.

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“…It has been shown that, in such a case, the His-tag does not interfere with the native protein structure. 28 The two molecules in the asymmetric unit of Nii4-WT were located side-by-side in identical orientations without any rotation. From the gel-filtration analysis, the molecular weight of the Nii4-WT was estimated to be 61.8 kDa.…”

Section: Structure Analysismentioning

confidence: 99%

Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

et al. 2012

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Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k cat /K m ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k cat ) and Michaelis constant (K m ) of Nii4-WT were lower than those of Nii3-WT. However, the k cat /K m of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k cat /K m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.

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His-tag impact on structure

Cited by 197 publications

References 18 publications

Multifunctional Properties of Glycoside Hydrolase Family 43 from Paenibacillus curdlanolyticus Strain B-6 Including Exo-β-xylosidase, Endo-xylanase, and α-L-Arabinofuranosidase Activities

Multifunctional Properties of Glycoside Hydrolase Family 43 from Paenibacillus curdlanolyticus Strain B-6 Including Exo-β-xylosidase, Endo-xylanase, and α-L-Arabinofuranosidase Activities

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

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