Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khorshid, H.R. Khoram; Samadi, Nasser; Khosroshahi, Shiva Ahdi; Jaliani, Hossein Zarei

doi:10.15171/apb.2015.084

Cited by 13 publications

(7 citation statements)

References 30 publications

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“…Using the natural potential of chaperones in their co-expression in host cells with target proteins which tend to misfold and aggregate in order to obtain their soluble forms has been demonstrated in Kim et al [10], where the production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, otherwise mainly expressed in inclusion bodies, was greatly enhanced by co-expression of folding accessory proteins, such as human peptidyl-prolyl cis-trans isomerase followed by co-expression of DnaK-DnaJ-GrpE together with GroEL-GroES. Similar results were reported for the expression in E. coli system of codon-optimized sarcosine oxidase from Thermomicrobium roseum, whose soluble expression was significantly enhanced via the co-expression of chaperones [11], for the expression of humanized single-chain antibody in E. coli [12], and others. Same strategy applies for the expression in yeast system, as, for instance, for the expression of xylose isomerase in Saccharomyces cerevisiae in functional state [13].…”

Section: Introductionsupporting

confidence: 82%

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

Федоров¹,

Yurkova²

2018

NanoWorld J

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Section: Introductionsupporting

confidence: 82%

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

Федоров¹,

Yurkova²

2018

NanoWorld J

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“…In addition, TF is related to GroEL, which can enhance the binding of GroEL substrate and promote protein folding or degradation (Kandror, Sherman, Moerschell, & Goldberg, 1997;Kandror, Sherman, Rhode, & Goldberg, 1995). The effectiveness of these chaperones on protein folding, stability, and aggregation has also been demonstrated (Maeng, Nam, & Kim, 2011;Nishihara, Kanemori, Kitagawa, Yanagi, & Yura, 1998;Veisi et al, 2015). In addition to chaperone proteins, fusion tagging technology also facilitates the expression of soluble proteins in E.coli .…”

Section: Discussionmentioning

confidence: 99%

Soluble Expression, Purification and Characterization of the VP3 of the 1st Serotype Bluetongue Virus

Wang

Han

Jia

et al. 2020

Preprint

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Bluetongue (BT) is a non-contact infectious disease of domestic or wild ruminants caused by the Bluetongue virus (BTV). It is transmitted by Culicoides biting midges. BTV mainly infect sheep, and animals infected with BTV usually show clinical symptoms such as fever, mucosal edema, and ulcers. BTV virus is an icosahedral symmetric RNA virus with 27 different serotypes. BTV consists of 7 structural proteins (VP1-VP7) and 4 non-structural proteins (NS1, NS2, NS3/NS3a, NS4, NS5). The VP3 encoded by the L3 gene is relatively high conserved structural protein, and constitute the inner symmetric icosahedral core shell of virus particle. In this study, VP3 recombinant protein of bluetongue I virus was successfully expressed and purified by prokaryotic expression system. Moreover, to increase the amount of soluble protein, we used chaperone protein ptf16 and two fusion proteins NusA and TRX. The results show that chaperone protein can increase the solubility of VP3. Then the expression conditions were optimized and VP3 was purified by nickel affinity chromatography. The VP3 immunize Balb/c mice and the results show that the serum titer is 1:1.28×105. In the Dot-ELISA assay, we found recombinant protein VP3 react with the serum from immunized mice by inactivate BTV-1. The results of IFA showed that the antibody produced by immunized mice with recombinant protein VP3 could react with the VP3 protein expressed in 293T cell. In conclusion, we expressed the recombinant protein VP3 with the same conformation as eukaryotic expression system and had same immunogenicity with inactivated virus, which laid a foundation for further study on the structure and function of BTV.

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“…The use of synthetic biology strategies to regulate the HSR of E. coli can help to avoid protein aggregation, increase protein solubility, and increase the yield of correctly folded recombinant target proteins. For example, overexpression of the heterologous protein in combination with chaperones (DnaK/DnaJ/GrpE, GroEL/GroES, ClpB) or s 32 proved to be effective in several studies because this enhanced protein folding, solubility, and activity [46,47]. IbpA and IbpB, two small HSPs, are also known to interact with the disaggregation and folding machinery of the cell [48].…”

Section: Environmental Applicationsmentioning

confidence: 99%

Potential Applications of the Escherichia coli Heat Shock Response in Synthetic Biology

Rodrigues

2018

Trends in Biotechnology

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The Escherichia coli heat shock response (HSR) is a complex mechanism triggered by heat shock and by a variety of other growth-impairing stresses. We explore here the potential use of the E. coli HSR mechanism in synthetic biology approaches. Several components of the regulatory mechanism (such as heat shock promoters, proteins, and RNA thermosensors) can be extremely valuable in the creation of a toolbox of well-characterized biological parts to construct biosensors or microbial cell factories with applications in the environment, industry, or healthcare. In the future, these systems can be used for instance to detect a pollutant in water, to regulate and optimize the production of a compound with industrial relevance, or to administer a therapeutic agent in vivo.

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Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

Abstract: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.

Cited by 13 publications

References 30 publications

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

Molecular Chaperone GroEL – toward a Nano Toolkit in Protein Engineering, Production and Pharmacy

Soluble Expression, Purification and Characterization of the VP3 of the 1st Serotype Bluetongue Virus

Potential Applications of the Escherichia coli Heat Shock Response in Synthetic Biology

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