Need more information: 1. Do you actually know the smaller protein is actually your target protein, or is it an impurity protein present in host cells? It could be that your truncation simply does not express well, and the darkest band in your crude lysate is something else. 2. Are you doing tag purification or are you purifying native protein? Purification conditions for truncated proteins may likely be very different than the native protein. Purification tags may stabilize your truncated protein (or not). 3. It is possible that the protein is being degraded in E. coli, but on the other hand I have made dozens of native N-terminal truncation variants without degradation. This is probably highly protein-dependent. 4. Does your truncation make chemical sense for protein stability? Is the N-terminus possibly required for the protein fold? Maybe another truncation site is appropriate, assuming you have a wild-type structure or homolog to go by. 5. It is possible to express your truncation variant as a protein-tagged (e.g. MBP or NusA, etc.) chimera, where you can control generation of the truncated variant after purification?
Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/21/2012 4:15 PM, Theresa Hsu wrote:
Dear all I made deletion mutation of a stretch of 20 amino acids on my protein. I can purify and crystallize wild type protein but not the mutated. Mass spec on gel separated protein shows degradation of mutant losing about another 150 amino acids. Is there any way of purifying this non-stable protein? I know nature has designed proteins to be stable. All steps are done at 4 C and protease inhibitor added during cell lysis for both proteins. Thank you.
