Thrombin is supposed to cleave the correctly sequenced and in-frame LVPR'GS site in the middle of a ~70kDa extracellular protein known to homotetramerize. The goal is to use the N-terminal MBP for expression and cut it off with thrombin prior to crystallizing the target protein, which is only a 25kDa fragment of a larger construct, but, even at 37ºC, thrombin fails to cleave it (determined by SDS-PAGE). I included my analysis of the possible causes, but I would like for someone who has experienced similar obstacles to tell me how he or she solved them. I've put way too many hours into designing a special vector and optimizing thrombin cleavage conditions to drop this project, so I'm open to any suggestions.

THROMBIN. The thrombin itself should not be an issue since, over a year ago, I successfully used many aliquots of the same batch, which, being frozen in the -80ºC in 2012, should have an almost indefinite shelf life.

HYDROPHOBIC INTERACTIONS. The cleavage site could be buried inside the construct's 3D conformation, rendering it inaccessible to thrombin. I've considered adding a mild detergent, such as a low concentration of SDS, to counteract that, but my PI said no.

REDOX. Since thrombin consists of two subunits bound by a disulfide bridge, it is super sensitive to reducing agents. While I've added no reducing agents to the mixture, the construct itself could have a redox ability. I wouldn't normally consider this due to the infinitesimally small probability; however, I've observed that, when I purify the construct from the lysate using nickel resin, the construct turns the nickel resin and elution product a faint greenish brown color, although, after elution, the resin returns to being good old nickel blue. Generally, color changes + nickel resin = some redox going on.