Recombinant Production of a Site-Specific Protease for Removal of Affinity Tags

Open Access
Nair, Abhinav C
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Joseph C. Reese, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
  • proteases
  • affinity tags
  • protein purification
  • TVMV
  • TEV
Protease enzymes are common in the natural world for their ability to cleave proteins, for example to digest food in the gut. The sequence specificity of certain proteases also makes them useful reagents to cleave affinity tags engineered into recombinant proteins. Tobacco Etch Virus (TEV) protease is widely used in modern biology work due to its high specificity and strong activity. Recent work shows that another protease, Tobacco Vein Mottling Virus (TVMV) protease, shares many of these desirable properties but with a different sequence specificity from TEV protease. The availability of two complementary proteases permits more affinity tags to be added to proteins and more sophisticated experiments to be designed. My goal in this project was to produce recombinant TVMV protease, to demonstrate robust protease activity, and to determine optimal protease reaction conditions. First a soluble version of the TVMV enzyme was created that utilized Maltose Binding Protein (MBP) as an N-terminal fusion tag. Protease activity was then assayed by protein digestion followed by SDS polyacrylamide gel electrophoresis. TVMV protease was found to be site-specific, and not cross-reactive with TEV protease, while demonstrating similar efficiency. Buffer type was not seen to significantly affect digestion and the TVMV enzyme was most active between room temperature and 30°C. Activity decreased with increasing salt concentration, becoming more pronounced at concentrations greater than 300 mM NaCl. Finally, the enzyme was expressed and purified on a larger scale to generate milligram quantities for future laboratory work.