problems with expression of mutant protein - (May/12/2006 )
I have problems with overexpression of a mutated human protein in Rosetta cells using 0.5 mM IPTG (final concentration) at 37 °C (3 hours induction).
The wild type is expressed well under the same conditions.
Prior sequencing of the vector revealed no frame shift or stop codon and I took care of using frequently used codons for the site directed mutagenesis.
Any ideas ?
Thank you very much for any advice,
What exactly is the problem, please? What kind of mutation have you introduced? Even though the wt expression is OK, have you tried changing any of the conditions - temp, [IPTG]? Have you tried/are you able to run a Western of the expression lysate to see if you're getting a truncation?
If all else is OK, try an in vitro expression. That way, you'll see if you are getting proteolysis in the cells.