How can cleavage of the fusion protein be slowed down during the purification process?
Purification should generally be carried out promptly in order to reduce the risk of in vitro cleavage. Both solutions and samples should be kept at 4°C. Although cell extracts should be loaded slowly onto the chitin column, the flow rate during the wash step can be increased. Some fusion proteins may cleave in vivo and during purification due to hydrolysis of the thioester linkage. If a significant portion of a fusion protein is cleaved in vivo, cleavage may occur during purification, resulting in lower final yield of the product. Since higher pH (>pH8) increases the rate of hydrolysis, extracts may be loaded and washed at pH6. On-column cleavage is then induced by equilibrating the chitin column using DTT-containing column buffer at pH8.
Purification should generally be carried out promptly and in the cold in order to reduce the risk of in vitro cleavage. Solutions and samples should be kept at 4°C. Although cell extracts should be loaded slowly onto the chitin column, the flow rate during the wash step can be increased. Some fusion proteins may cleave in vivo and during purification due to hydrolysis of the thioester linkage. If a significant portion of a fusion protein is cleaved in vivo, cleavage may occur during purification, resulting in lower final yield of the product. Rarely does a significant amount of cleavage occur during purification; since higher pH (>pH8) increases the rate of hydrolysis.
