Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System

Zhang, Jian; Sun, Aiyou; Dong, Yi; Wei, Dongzhi

doi:10.1007/s12010-017-2599-9

Cited by 11 publications

(5 citation statements)

References 34 publications

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“…However, the overly extended sequence in front of the target protein could remain a persistent concern for its future applications and in most cases, it is required to be removed even though it does greatly enhance the expression (Zhang et al 2018). This extra step will add uncertainties to the final production for the recombinant protein.…”

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

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“…Since the only difference between both vectors is in the presence or absence of S-Tag, the result consistently confirmed that the S-Tag had critical influence on the expression of iLRP as expected. However, the overly extended sequence in front of the target protein could remain a persistent concern for its future applications and in most cases, it is required to be removed even though it does greatly enhance the expression [20]. This extra step will add uncertainties to the final production for the recombinant protein.…”

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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“…The premature cleavage of the fusion tag, as observed with the autocatalytic intein 10 , is completely avoided by the requirement of an external protease. While the SUMO strategy is nowadays widely used for recombinant protein production 31 32 33 , we demonstrate in this paper that it is especially useful for the generation of an intrinsically disordered, aggregation-prone, amyloidogenic protein such as Httex1. We believe that the simplicity, efficiency and robustness of our SUMO-fusion-based method will make native, tag-free Httex1 more accessible to researchers from different disciplines and eliminate the need to use non-native sequences of Httex1 in vitro .…”

Section: Introductionmentioning

confidence: 85%

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Reif

Chiki

Ricci

et al. 2018

JoVE

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Huntington's Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (≥36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (≥42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

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Recombinant Production and Characterization of SAC, the Core Domain of Par-4, by SUMO Fusion System

Cited by 11 publications

References 34 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

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

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