After inserting the gene in pet 22b vector, I got it sequenced with good results and initiated expression in rosetta cells. After the OD reached 0.6 induced expression with 1mM IPTG and incubated at 16 deg for 16 hrs as described by a paper which has followed the same protocol for its crystallization. But the problem is, the uninduced is showing bands but the induced is not showing any bands on PAGE.
Please suggest any troubleshooting methods.
You can check your induction at 2,4,6,8 hrs aliquotes,may be protein was degraded at the early stages of expression and reduce the conc. of IPTG as low as possible and also check whether your protein have auto catalytic activity or not ?
Repeat exactly as described in the paper - same vector/construct, same strain. You also can ask the corresponding author of that paper to send the construct, they must.
thank you all for ur advices,
@ivan i am doing exactly as described in the paper with only a difference the authors used the rosetta (DE3) PlysS i am using rosetta2 (DE3) PlysS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833039/
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