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Protein Overexpression
Exploring Mutant Proteins
Analysis of mutations
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You sequence all the mutant proteins from the rare fluorescent colonies. This gives you the identities of the amino acid changes that leads to moderate decreases in precipitation. If you could understand why these changes work, then perhaps you could figure out how to make even more effective changes, either in other amino acids or by combining mutations.
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Wild-type UDP-glc DH
...SKVDKINNGLSPIQDEYIEY...
Mutant UDP-glc DH
...SKVEKINNGLSPIQDEYIEY...
...SKVDKINNGVSPIQDEYIEY...
...SKVDKINNGLSPIQDESIEY...
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You wish you could visualize where on the protein's three dimensional structure the effective mutations are situated, for then you might learn which other amino acids in the vicinity are candidates for directed mutagenesis. This way you might rationally design a protein that won't precipitate.
There IS a three-dimensional structure known for a related protein, UDP glucose dehydrogenase from Streptococcus pyogenes, but nothing structural is known about YOUR protein. Tough luck?
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