title: Cleavage of fusion proteins on the affinity resins using the TEV protease variant.
abstract: It is documented that the tobacco etch virus protease (TEVp) variant TEVp3Mis less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp5Mvariant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp3M. The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp5Mat specified conditions using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp5Mconstructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp5Mefficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp5Mmediating tag removal enabled purification and cleavage of the fusion proteins, removal of the protease, and separation of the target proteins from the affinity resin to be accomplished in one step.
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Kejun
authors.initials: K
authors.lastname: Zhu
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Yan
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Mo
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Xie
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Beijiu
authors.initials: B
authors.lastname: Cheng
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China. Electronic address: fanjun@ahau.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
mesh_terms: D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease
keywords: Affinity resins; Fusion tags; On-resin cleavage; Purification; Tobacco etch virus protease