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Applied Biochemistry and Biotechnology

, Volume 182, Issue 2, pp 769–781 | Cite as

Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli

  • Xuelian Yu
  • Jiaqi Sun
  • Weiyu Wang
  • Li Jiang
  • Beijiu Cheng
  • Jun FanEmail author
Article

Abstract

In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

Keywords

Fusion tags TEV protease Target proteins Soluble production Escherichia coli 

Notes

Acknowledgments

This work is supported by the Scientific and Technological Project of Anhui Province (1506c085007).

Supplementary material

12010_2016_2360_MOESM1_ESM.tif (2.2 mb)
Figure S1 SDS-PAGE analysis of the fusion protein cleaved by the TEVp constructs extracted from the recombinant E. coli BL21(DE3) cells (a) and Rosetta™ (DE3) cells (b). M: protein marker. CK: the protein substrate is incubated with the inactive TEVpC151A variant. The fusion protein substrate and the cleaved products are indicated by arrows. (TIFF 2262 kb)
12010_2016_2360_Fig7_ESM.gif (6 kb)

High Resolution Image (GIF 6 kb)

12010_2016_2360_MOESM2_ESM.tif (4.1 mb)
Figure S2 The pigmented E. coli cells overexpressing the fusions proteins for EcGTR. Recombinant cells carrying pET-28b plasmids are used as the control. (TIFF 4194 kb)
12010_2016_2360_Fig8_ESM.gif (28 kb)

High Resolution Image (GIF 28 kb)

12010_2016_2360_MOESM3_ESM.tif (1.9 mb)
Figure S3 SDS-PAGE analysis of the purified Ex-TEVpH6 and MBP-TEVpH6 proteins by Ni-NTA agarose. M: Protein marker. (TIFF 1971 kb)
12010_2016_2360_Fig9_ESM.gif (4 kb)

High Resolution Image (GIF 4 kb)

12010_2016_2360_MOESM4_ESM.doc (33 kb)
Table S1 Primers used in this study (DOC 33 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Key Laboratory of Crop Biology of Anhui ProvinceAnhui Agricultural UniversityHefeiPeople’s Republic of China

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