It is documented that the tobacco etch virus protease (TEVp) variant TEVp(3M) is 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 TEVp(5M) variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp(3M). The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp(5M) at specified conditions using GFP reporter for visual track and detection. After on column cleavage of three fusion constructs using the cognate TEVp(5M) constructs, 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 TEVp(5M) efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp(5M) mediating 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. (C) 2016 Elsevier Inc. All rights reserved.