What are the solutions to reduce co-purification of CBD fusion proteins?
In some experiments, uncleaved fusion protein and intein-CBD species may be present in various amounts in the elution samples. This leakage may occur, for example, when the chitin column is saturated. Furthermore, when cleavage is inefficient, the fusion protein may appear as a dominant contamination species. Several approaches may be used to improve the purity of the eluted samples. First, an increase in the chitin column volume may help to reduce potential overloading problems. Also make sure that the load is dilute (resuspend pellet from 1L of cells in at least 100 mls Column buffer). Second, use of column buffer without detergents may help to reduce the background in some cases. Third, wask the column with at least 20 column volumes of buffer. Finally, the CBD fusion proteins may be reduced by passage of the elution sample slowly through a second chitin column. To improve cleavage efficiency a different fusion construct, for example, with additional favorable residue(s) adjacent to
