Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

Zhao, Dongxin; Huang, Zhong‐Xian

doi:10.1155/2016/8206854

Cited by 46 publications

(26 citation statements)

References 30 publications

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“…The tag is not only simple in structure composing of only 6 histidines with no need to remove it from the final products, but also very convenient for the follow-up research on the engineered protein. The label has strong affinity with Ni + , which makes the purification of overexpressed proteins simple by using Ni + -affinity column (Zhao and Huang 2016). In addition, the expressed products can be easily verified and detected by the monoclonal antibody against 6xHis-Tag that is very common in the current market (Yin et al 2014).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Liu

Dong

Yan

et al. 2020

Preprint

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iLRP (immature laminin receptor protein) is a tumor associated antigen over-expressed on the surface of most human cancer cells and plays an important role in the process of tumorigenesis and development. It has strong auto-immunogenicity in patients and is a good target protein for tumor immunotherapy. To find a practical and an efficient method for the production of recombinant iLRP, three expression vectors based on pET-30a(+) from pET expression systems were constructed. The first one is to add a 6xHis-Tag to the N-terminal of natural iLRP, which is called as pET-His-iLRP. The second one is to include an S-Tag between the C-terminal of 6xHis-Tag and the N-terminal of natural iLRP, which is called as pET-His-S-iLRP. The third one only contains the 6xHis-Tag in front of the N-terminal of iLRP, but the natural iLRP gene was first optimized according to the bacterial genome, and the constructed vector was named as pET-His-Opt-iLRP. Then the expression of three vectors in Escherichia coli (E. coli) BL21 (DE3) was analyzed. Results demonstrated that the iLRP expression was the highest in pET-His-Opt-iLRP, followed by pET-His-S-iLRP, and pET-His-iLRP expressed the lowest iLRP. This implied that the vector combining the codon-optimized iLRP gene with a 6xHis-Tag had the best effect on the expression of iLRP, and the expression in vectors with the natural iLRP gene depends on its leader sequences in the expression frame. It can be consequently concluded that the heterologous expression of human iLRP in a bacterial host could be greatly increased by genetically modifying gene constituents and using proper protein-tags, which laying the foundation on the future researches on its applications.

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Section: Discussionmentioning

confidence: 99%

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Liu

Dong

Yan

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…The tag is not only simple in structure composing of only 6 histidines with no need to remove it from the final products, but also very convenient for the follow-up research on the engineered protein. The label has strong affinity with Ni + , which makes the purification of overexpressed proteins simple by using Ni + -affinity column [14]. In addition, the expressed products can be easily verified and detected by the monoclonal antibody against 6xHis-Tag that is very common in the current market [15].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Liu

Zhang

Yan

et al. 2019

Preprint

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Background: iLRP (immature laminin receptor protein) is a tumor associated antigen over-expressed on the surface of most human cancer cells and plays an important role in the process of tumorigenesis and development. It has strong auto-immunogenicity in patients and is a good target protein for tumor immunotherapy. The aim of this study is to find a practical and an efficient method for the production of recombinant iLRP, and to provide enough recombinant protein for further study of its potential applications.Results: In this report, three different expression vectors based on pET-30a(+) from pET expression systems were constructed. The first one is to add a 6xHis-Tag to the N-terminal of natural iLRP, which is called as pET-His-iLRP. The second one is to include an S-Tag between the C-terminal of 6xHis-Tag and the N-terminal of natural iLRP, which is called as pET-His-S-iLRP. The third one only contains the 6xHis-Tag in front of the N-terminal of iLRP, but the natural iLRP gene was first optimized according to the bacterial genome, and the constructed vector was named as pET-His-Opt-iLRP. Then the expression of three vectors in Escherichia coli (E. coli) BL21 (DE3) was analyzed. Results demonstrated that the expression of iLRP was the highest in the vector of pET-His-Opt-iLRP, followed by the vector of pET-His-S-iLRP, and the vector of pET-His-iLRP expressed the lowest iLRP.Conclusions: According to the results, it was concluded that the vetor pET-His-Opt-iLRP combining the codon-optimized iLRP gene with a 6xHis-Tag had the best effect on the expression of iLRP, and the expression in vectors with the natural iLRP gene depends on its leader sequences in the expression frame. The longer leader sequences accommodate to the host E. coli, the higher expression the engineered gene would be.

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“…In comparison with the natural keratinase KerA purified from Bacillus licheniformis (6000 U/mg), weaker activity was observed for heterologous expression in insect cells of rKer. That could be as a result of the addition of purified tag 8His-Flag [ 23 , 24 ]. However, the keratinalytic activity of recombinants from AcMPNV-Ker-His-Flag/Sf9 was higher than those of previous study using E .…”

Section: Discussionmentioning

confidence: 99%

Secretory expression and purification of Bacillus licheniformis keratinase in insect cells

Huang¹,

Chen

Ren³

2017

PLoS ONE

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The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ cell to generate recombinant bacmid carrying Ker-His-Flag. Recombinant viruses were produced by infecting insect Spodoptera frugiperda (Sf9) cells with bacmid DNA and used for proteins production. Target proteins were purified from the cell supernatants by Ni2+-NTA affinity chromatography and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The purified product contained two peptides with molecular weights of 38 kDa and 30 kDa and had an optimal pH and temperature at 8.0 and 45°C for keratinolytic activity, respectively. The final product had a specific activity of about 635 U/mg. In summary, we have demonstrated that the open reading frame containing recombinant Ker-His-Flag was expressed and secreted by leader peptide of mellittin from Apis mellitera in insect cells and affinity purification through 8His-Flag tag. It presents an alternative technology for producing keratinases. To our knowledge, it was the first report on the expression of functional keratinase from Bacillus licheniformis in insect cells system.

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Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

Cited by 46 publications

References 30 publications

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Evaluation of three different vectors on human iLRP expression in Escherichia coli

Secretory expression and purification of Bacillus licheniformis keratinase in insect cells

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