Recombinant protein expression

[Susheel K Sharma]

Hi

I have a problem in the expression of plant viral gene in E. coli system. The gene is cloned in pET28a and the sequence is in frame (confirmed by sequencing). I have tried the expreesion in DE3 (BL21), PlysS, Rosette PlysS, but could not get the expression. The expected sixe of protein is 48 KDa and 14 KDa. Kindly suggest me some troubleshoot.

Edited April 8, 2012 by Susheel K Sharma

[chance.lee]

In general, there are two reasons.

One is the expression vector problem, and another problem is receptor expression. Vector may be mutated or modified to lead promoter deletion or location changes. In respect of receptor, receptor mutations can also cause no-expression. In some cases, certain genes may not be suitable for expression in certain receptors. in some cases, even if both are correct expression, you may also need to look more screening, such as:

1. always transform fresh - this is really a must do
2. always have glycerol stocks of cloning (not expressing) strains - another must do. I particularly like XL-1 Blue
3. I found carbenicillin much better than Amp - and also ensure the antibiotic stocks are well kept. If in doubt try another stock.
4. if nothing works, we found that for very old stocks (a few years old) sometimes the best option is just go back to PCR and reinsert in fresh plasmid. At times you may get 1 single mutation which is difficult to detect, but it will heavily affect expression.
5. of course induction times and conditions are important too, but my guess is they will affect only the yield - you should still see some protein. IPTG stocks should also be well kept.

More question about protein expression in e coli, you can refer to.

[Justin.Frank]

why do people want to finish all the thing their self? these experiment can be performed by commercial companies rapidly, such as the following one: creative-biostructure.com.