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mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli

1
KAIST Institute for BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
2
NTT Hi-Tech Institute, Nguyen Tat Thanh University (NTTU), Ho Chi Minh City 700000, Vietnam
3
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
4
Biology Department, Science and Technology Faculty, Universitas Airlangga Mulyorejo, Surabaya 60115, Indonesia
5
Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), Ulsan 44429, Korea
6
Intelligent Synthetic Biology Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(13), 3163; https://doi.org/10.3390/ijms20133163
Received: 6 June 2019 / Revised: 18 June 2019 / Accepted: 21 June 2019 / Published: 28 June 2019
(This article belongs to the Special Issue Molecular Chaperones 2.0)
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Abstract

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3ʹ untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5ʹ-TAATG-3ʹ between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes. View Full-Text
Keywords: molecular chaperone; DnaJK-GrpE; co-translational folding; RNA scaffold; two-cistron expression; inclusion body; recombinant protein; protein aggregation; mRNA engineering; protein solubilization molecular chaperone; DnaJK-GrpE; co-translational folding; RNA scaffold; two-cistron expression; inclusion body; recombinant protein; protein aggregation; mRNA engineering; protein solubilization
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Bui, L.M.; Geraldi, A.; Nguyen, T.T.; Lee, J.H.; Lee, J.Y.; Cho, B.-K.; Kim, S.C. mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli. Int. J. Mol. Sci. 2019, 20, 3163.

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