Dear Kris, the release of the desired protein from a fusion protein is affected by the adjacent amino acid sequences (e.g. the presence of a Proline after Lys makes the protease activity unworkable) at the cleavage site as well as by the size of the two fused components and of course by the accessibility of the cleavage site (the protein folding could bury the cleavage site). Moreover, did you try different enzyme/substrate ratio and different time of incubation to find out suitable conditions? Another thing I know is that high concentration of salt such as NaCl inhibits the cleavage reaction, as well the presence of unsoluble aggregates. I would suggest to perform a dialysis step to remove any salt if you have and dilute the fusion protein to 0.2-0.5 mg/ml. Which kind of buffer are you using in the purification process? Phosphate buffer reduces the activity of the enzyme. Finally if it doesn't work changing these conditions, I would suggest to use a proper concentration of denaturing agents (low concentration) to slacken the fusion protein structure and to increase then the accessibility of the cleavage site (if it is the case of a buried cleavage site).
Best, Mirella Vivoli Mirella Postdoctoral Fellow University of Exeter, Biosciences Biocatalysis Centre, Henry Wellcome Building Stocker Road, Exeter, Ex4 4QD Tel:+ 44 (0)1392 726121 ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of K Singh [ksc...@gmail.com] Sent: Sunday, July 21, 2013 8:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] problems with cleaving protein Dear All, I have expressed fusion protein that is expressed in the following format Fusion partner - Enterokinase cleavage site - Protein of interest. somehow enterokinase is not doing its job though following manufacturers recommendations. any inputs like what protocol worked. Many thanks for your inputs Kris
