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an imperfect site on a flexible loop will Diagnose before redesigning : intact-mass LC-MS plus N-terminal sequencing of the unwanted fragment localizes the cryptic cut to within ~1 residue Mitigation is usually a condition change, not a sequence change : cold-temperature, on-column cleavage, and reduced enzyme:substrate ratio rescue 70%+ of cryptic-cleavage problems before mutagenesis is needed The Problem Most Labs Pretend Doesn't Exist Open any protein production paper and you will read sentences like "the tag was removed by overnight digestion with TEV protease." What you won't read is "the protease also clipped an internal loop at residue 187, reducing yield by 40%, but we cropped the gel." The literature massively under-reports cryptic proteolysis because the failure modes are private they live in lab notebooks, not papers

[Google Scholar] 99.Dahlstroem A., Fuxe K