Cannot get any protein overexpression in Rosetta? - (Mar/26/2007 )

Hie,

I've been trying to overexpress a his-tagged recombinant protein in E.Coli Rosetta strain with the pet28b vector (Recombinant protein is adh from Sulfolobus). I've checked the sequence of the insert and verified them by restriction digest. I failed to overexpress the protein even after several attempts. I grow the cultures overnight in 2mL LB + kanamycin + chloramphenicol from a frozen. And then innoculate 50mL cultures the following day with the appropriate antibiotic. I induce the cultures when they reach an OD of 0.4 - 0.5. I've tried inducing the protein with both 0.5mM of IPTG & 1 mM IPTG, growing the cultures at 37 degrees and 30 degrees, induction period of 3 & 6 hours. Then I check both the soluble and insoluble fraction by SDS Page.


I'm new to this and I've run out of ideas to fix it. Any ideas of what could be the problem will be greatly appreciated.

Thanks

Hi there,
I think you should make a survey to find out the optimal expression condition for your target protein. Parameters could affect the expression level include: induction time (2,4,6,8,12,24h), temperature for induction: 37, 30, 25, 20. and when you put your cultures in the temperature for induction, let them at low shaking in order to your strain adapt to the temperature (10-15mins). Moreover, how long do your strains grow up OD 0.4-0.5? It's better to take about 1h to get that OD means that your strain is strong and fresh to express. If not, reconsider your overnight activation condition. And you should make a Western-Blot to check the expression of your target protein (yes/no?)
Good luck!