Hello,

My question is two-fold, first a lysis problem and then a separation problem. My apologies for the unavoidably lengthy nature of the question:

Our protocol is to obtain E. coli inclusion bodies using Novagen's pET vector and Autoinduction system (works wonderfully), then lysing the cells with BugBuster (detergent solution with lyosozyme and benzonase to digest DNA).

With one particular 8 kDa peptide (natively unfolded, pI ~11, amyloidogenic) these lysis conditions result in the cleavage of the last 6 amino acids, shown by MALDI, such that the resulting inclusion bodies are a mixture of the intact peptide and the C-terminally cleaved derivative. Protease inhibitors don't help. Sonication is also damaging. The addition of at least 25mM EDTA is prophylactic but abolishes any benzonase or DNaseI activity, making the inclusion bodies difficult to pellet.

My questions are: a) whether anyone has this metal-facilitated cleavage problem during lysis and how it was solved and b) should I expect to achieve a good separation of these two peptides by HPLC (~8227 Da vs. 7750 Da) cation exchange or RP-HPLC?

Note, I use an AKTA low pressure LC system quite often and these cation exchange resins (CM sepharose, SP sepharose, Resource S) bind beautifully but do not give enough resolution separate the two peptides which only differ by 6 amino acids. The truncated peptide is useless for our research.... Thanks,

Roger