If the fusion protein forms inclusion bodies, can I try to purify protein under denaturing conditions?
Purification and cleavage of the C-terminal fusion (MBP-Sce VMA intein-CBD fusion) protein has been tested under various urea concentrations. 1-4 M urea allowed 80% binding efficiency while 5-8 M urea showed 30-50% binding efficiency. The efficiency of cleavage with 30 mM DTT in 2 M urea is at least 50%, in comparison to reactions under native conditions. Based on these data, chitin chromatography and on-column cleavage may be carried out in 1-2 M urea. Since DTT may cause rapid cleavage of the fusion protein under denaturing conditions, following the wash step the column flow should be stopped and the DTT stock solution should be added directly to the column.