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Bioprocess and Biosystems Engineering

, Volume 40, Issue 7, pp 1101–1110 | Cite as

Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose

  • Xuelian Yu
  • Jiaqi Sun
  • Weiyu Wang
  • Li Jiang
  • Ruyue Wang
  • Wenjun Xiao
  • Beijiu Cheng
  • Jun FanEmail author
Research Paper

Abstract

In this study, four fusion proteins were designed, in which the N-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (RAC), and the release of the C-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (TEVp) variant, or the immobilized cognate protease with a binding capacity of up to 220 mg protein per gram of RAC. The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate. On-resin cleavage and purity of the released target proteins are related to the context of the fusion tag, the incorporated linker composition, and the colored protein. Owing to low cost and high binding capacity of the RAC, the TEVp immobilized on the resin is an ideal alternative for removing fusion tag. The colored proteins are easily monitored in the on-resin process of fusion proteins, and rapid separation from RAC.

Keywords

TEV protease variant Oriented immobilization Regenerated amorphous cellulose On-resin cleavage Visual detection 

Notes

Acknowledgements

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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

449_2017_1772_MOESM1_ESM.tif (1.8 mb)
Figure S1. Purification of CBM-TEVpH6. The eluent from Ni–NTA agarose or proteins bound to RAC are mixed with SDS-PAGE loading buffer, boiled for 10 min, centrifuged, and analyzed. M: protein molecular weight marker. Lane 1: the eluent from Ni–NTA agarose. Lane 2: proteins bound to RAC. Arrow indicates the position of CBM-TEVpH6 (TIFF 1873 kb)
449_2017_1772_MOESM2_ESM.tif (4.3 mb)
Figure S2 Time course cleavage of GST-tevS-eDAL with the immobilized or free TEVp construct. After cleavage, the mixture is analyzed by SDS-PAGE. a Cleavage of fusion protein substrate with the immobilized TEVp construct. b Cleavage of fusion protein substrate with the free TEVp construct. M: protein molecular weight marker. The purified fusion protein and/or cleaved products are indicated by arrows. Incubation time is denoted on the top of the gel. c Activity of the released eDAL (TIFF 4423 kb)
449_2017_1772_MOESM3_ESM.tif (5.2 mb)
Figure S3 The CBM-tevS-VHb bound RAC with incubation of the inactive and active TEVp constructs. a Visual detection of the fusion protein incubated the protease constructs. b Visual detection of the RAC matrices and eluents treated with the proteases. c SDS-PAGE analysis. M: protein molecular weight marker. All of resins, eluents, and proteins on the SDS-PAGE gel, are treated correspondently as CBM-2tevS-VHb (TIFF 5335 kb)
449_2017_1772_MOESM4_ESM.tif (7.1 mb)
Figure S4 Detection of the introduced flexible linker on improving cleavage efficiency of the fusion protein GST-tevS-ZmSR bound to glutathione Sepharose 4B. a Cleavage in solution analysis. M: protein molecular weight marker. Lane 1: purified GST-tevS-ZmSR, Lane 2: the fusion protein substrate is cleaved in solution. b On-resin cleavage of the fusion protein with the tevS. Lane 1: purified GST-tevS-ZmSR. Lane 2: eluents after on-resin cleavage. Lane 3: the proteins bound to the affinity resin. c On-resin cleavage of the fusion protein with incorporation of the H6D4 linker between GST and tevS. The protein sample in each lane is corresponded with the figure S4 b (TIFF 7250 kb)
449_2017_1772_MOESM5_ESM.tif (2.4 mb)
Figure S5 Incubation of the CBM-tevS-VHb in crude extracts with the soluble CBM-TEVpH6 construct. The mass ratio of the fusion protein amount in crude extracts by determined Bradford method to the soluble TEVp construct purified by Ni–NTA resin is 5:1. The mixture is reacted at 30 °C for 3 h and analyzed by 15% SDS-PAGE. M: protein molecular weight marker. Arrows indicate the TEVp constructs, the fusion protein substrate and cleaved products. Lane 1: the fusion protein incubated with the TEVpC151A construct. Lane 2: the fusion protein incubated with the active TEVp construct (TIFF 2495 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

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

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