hi to all, I am currently dealing with a cysteine rich protein having a cysteine metal binding domain cxxc and another cysteine rich motif that could be a heavy metal binding domain CxxxCxxCxC. the protein is his tagged and I can't modify the sequence. the problem is that when I examine the e.coli extracts, the protein is unfolded and aggregates.
any advices?
What part of the bacteria are you purifying it from? i.e. Cytoplasm or periplasm? Do you have an idea what metal it binds? You may consider adding a periplasmic expression signal peptide to the N-terminus, such as the PelB sequence and add the suspected metal to the culture media. The metal could provide significant stability during folding. You can extract the periplasmic fraction by osmotic shock with lysozyme. Also you may consider modifying your expression conditions, such as inducer concentration, temperature, and time, if you haven't done so already.
@Rosato
What is your buffer condition? Did you maintain a reduced environment in the buffer by adding sufficient amount of DTT or b-mercaptoethanol. You need to optimize the concentration of DTT or b-mercaptoethanol with a prior knowledge of the activity of the thiol groups of your protein.
try adding 20 mM betamercaptoethanol when break cells and during Ni-NTA purification
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