MRNA engineering for the efficient chaperone-mediated co-translational folding of recombinant proteins in escherichia coli

Le Minh Bui, Almando Geraldi, Thi Thuy Nguyen, Jun Hyoung Lee, Ju Young Lee, Byung Kwan Cho, Sun Chang Kim

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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 3’ 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 5’-TAATG-3’ 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 effiective tools for producing soluble therapeutic proteins and enzymes.

Original languageEnglish
Article number3163
JournalInternational Journal of Molecular Sciences
Volume20
Issue number13
DOIs
Publication statusPublished - 1 Jul 2019

Keywords

  • Co-translational folding
  • DnaJK-GrpE
  • Inclusion body
  • MRNA engineering
  • Molecular chaperone
  • Protein aggregation
  • Protein solubilization
  • RNA scaffiold
  • Recombinant protein
  • Two-cistron expression

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