Cleavage of Fusion Proteins
Conditions: Because of the subtle changes in secondary and/or tertiary structure which occur among different fusion proteins we cannot recommend a precise set of conditions that will optimize the factor Xa or thrombin cleavage of all fusion proteins. Instead, we offer some basic recommendations for starting conditions, and then suggest changes that may optimize the cleavage of your protein.
Enzymes: Bovine factor Xa (HTI catalog #BCXA-1060) is employed for removal of affinity tags from fusion proteins which contain a factor Xa recognition site. Human alpha-thrombin (HTI catalog #HCT-0020) is employed for removal of affinity tags from fusion proteins which contain a thrombin recognition site.
Concentrations: Start with a 1:50 molar ratio of enzyme to substrate (this is assuming that your substrate protein is in solution in the range of 1 to 50 micromolar). To make this calculation, you can use a molecular weight of 45,300 for bovine factor Xa, and 36,700 for both human or bovine thrombin.
Buffer choices: Thrombin and factor Xa are serine proteases that function best when the pH is between 7.0 and 8.5, and the ionic strength is near or equivalent to that of a 0.15M NaCl solution. TBS or HBS (20mM Tris (or Hepes), pH 7.5, containing 0.15M NaCl) are suitable buffers. For factor Xa, we also recommend including 2 mM CaCl2.
Temperature: Generally these experiments are done at room temperature, although elevated temperatures (i.e., 37oC) which increase the hydrolysis rate will also work.
Optimization: To optimize the cleavage of fusion proteins a time-course experiment followed by SDS-PAGE analysis is often useful. Data from the time course experiment should be examined for both completeness of cleavage, as well as specificity. Generally reactions can be accelerated by increasing: a) the enzyme concentration; b) the temperature; and c) the pH (but not over pH 8.5), however an increased rate may also be accompanied by non-specific cleavage patterns.