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

Федоров, А. Н.; Yurkova, Maria S.

doi:10.17756/nwj.2018-053

Cited by 7 publications

(2 citation statements)

References 64 publications

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“…The main purpose of our work was to develop a widely applicable fusion protein system that would be specifically suitable for the production of different peptides which are difficult to express in bacteria for the reasons described above, and that would also allow simple, robust and scalable purification procedures of target polypeptides. The chaperone GroEL is a fitting candidate to be used as a carrier in such a fusion system 18 . It comprises a large particle consisting of two heptamers with a large cavity inside, which accommodates the protein substrate.…”

Section: Introductionmentioning

confidence: 99%

Production of a toxic polypeptide as a fusion inside GroEL cavity

Yurkova

Садыхов

Федоров

2020

Sci Rep

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The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.

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Section: Introductionmentioning

confidence: 99%

Production of a toxic polypeptide as a fusion inside GroEL cavity

Yurkova

Садыхов

Федоров

2020

Sci Rep

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“…Co-expression of endoplasmic reticulum chaperones was also used to increase secretion level of the class II hydrophobin HFBI in Pichia pastoris 16 . The use of bacterial chaperones for stabilization of target proteins in different stages of biosynthetic production is reviewed previously in 17 . Still, with all the current progress and achievements in protein fusion technologies, there are many proteins of interest for research and biotechnology which are partly or completely insoluble by themselves and it would be beneficial to improve their solubility.…”

Section: Introductionmentioning

confidence: 99%

Versatile format of minichaperone-based protein fusion system

Yurkova

Sharapova

Zenin

et al. 2019

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Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

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