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Versatile format of minichaperone-based protein fusion system.

ABSTRACT: 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.

SUBMITTER: Yurkova MS

PROVIDER: S-EPMC6803692 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Versatile format of minichaperone-based protein fusion system.

Yurkova Maria S MS Sharapova Olga A OA Zenin Vladimir A VA Fedorov Alexey N AN

Scientific reports 20191021 1

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 ...[more]

PMID: 31636289

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Project description:Nanobodies show a great potential in biomedical and biotechnology applications. Bacterial expression is the most widely used expression system for nanobody production. However, the yield of nanobodies is relatively low compared to that of eukaryotic systems. In this study, the repetitive amino acid sequence motifs (GAGAGS) found in silk fibroin protein (SFP) were developed as a novel fusion tag (SF-tag) to enhance the expression of nanobodies in Escherichia coli. SF-tags of 1 to 5 hexapeptide units were fused to the C-terminus of 4G8, a nanobody against human epididymis protein 4 (HE4). The protein yield of 4G8 variants was increased by the extension of hexapeptide units and achieved a 2.5 ~ 7.1-fold increase compared with that of untagged 4G8 (protein yield of 4G8-5C = 0.307 mg/g vs that of untagged 4G8 = 0.043 mg/g). Moreover, the fusion of SF-tags not only had no significant effect on the affinity of 4G8, but also showed a slight increase in the thermal stability of SF-tag-fused 4G8 variants. The fusion of SF-tags increased the transcription of 4G8 by 2.3 ~ 7.0-fold, indicating SF-tags enhanced the protein expression at the transcriptional level. To verify the applicability of the SF-tags for other nanobody expression, we further investigated the protein expression of two other anti-HE4 nanobodies 1G8 and 3A3 upon fusion with the SF-tags. Results indicated that the SF-tags enhanced the protein expression up to 5.2-fold and 5.7-fold for 1G8 and 3A3, respectively. For the first time, this study reported a novel and versatile fusion tag system based on the SFP for improving nanobody expression in Escherichia coli, which may enhance its potential for wider applications.Key points• A silk fibroin protein-based fusion tag (SF-tag) was developed to enhance the expression of nanobodies in Escherichia coli.• The SF-tag enhanced the nanobody expression at the transcriptional level.• The fusion of SF-tag had no significant effect on the affinity of nanobodies and could slightly increase the thermal stability of nanobodies.

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Versatile format of minichaperone-based protein fusion system.

Publications

Versatile format of minichaperone-based protein fusion system.

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