more specific questions:

1) Does chloramphenicol really need to be added?

2) Is the medium for protein expression general LB/Amp?

3) Is IPTG for inducing expression ?

4) Is the temperature for culturing at 37C or 30C?

It would be very grateful if anybody can help with the questions.

Thanks a lot

1) Yes. You need it as I recall for the tRNAs that are in the rosetta strain

2) Yes. Try also 2XLB and 100 to 300mg amp

3) Yes. However, this is not a T7 lac promoter so you may require less ie 0.3mM.

You should try a range of [IPTG]

4) For overnight expression or unfolded proteins the temp. should be lower ie 20-30C

For expression of a few hours you can use 37C

Many thanks, Ted. I shall try your suggestions.

You need to add chloramphenicol along with ampicillin.

You can try different media for optimization of expression levels. You can try LB and TB media.

IPTG for Rosetta strain, you can use 100uM concentration.

You can decrease the induction temp to 18 - 20C . Induce the culture overnight.

I generally use TB with 100uM IPTG and 18C induction temp which worked very well for most of my experiments.

Thanks everyone again! I have tried the above suggestions, here are the results and new problem:

1) From SDS-PAGE, it sounds there is band which the size is equivalent to the target protein. However it can be seen in the negative un-induced samples (no IPTG added) but the density is slightly lower.

I wonder if I need to optimize the medium, e.g adding glucose etc to repress the expression. Does anyone can suggest a good idea please?

Many thanks for your help.