What restriction enzyme did you use in the forward and reverse primer?

The forward was NdeI and reverse was BamHI.

Hello Mahbobeh,

which bacterial strain did you transform to perform the expression? Is there any literature describing expression of this protein or related proteins previously?

Does the protein induce at all using IPTG at 37˚C in a short incubation following induction (i.e., 1 hour, 2 hours etc.)? Unless the protein is known to be difficult to induce at all, I would always start verifying expression using a shorter induction at 37˚C to see a band, then think about longer cooler inductions to maximise soluble expression once one has established that the vector / cell combination give protein when induced.

Feel free to keep asking questions until we can come up with a solution. Best of luck! Robin

Thanks Mangal for the answer.

the bacterial strain was BL21 codon plus. Yes, there are literature on this and it should not be a difficult protein. I actually used 2hr induction but there was not much difference in banding pattern there either. I mean with overnight induction many more protein were there but i couldn't see the difference between induced and in-induced one except that only an 11kDa was in induced one, which was always there.

I will do western to see if it's the truncated protein or not.

I am also doubtful on my clone. Will re-sequence it to see what comes out.

The clone has been very difficult when re-culturing from the bacterial stock. I think i maybe have an instable clone. Then what i should do?

Best,

Mahbobeh

Thanks Robin for the sugeesion.

the bacterial strain was BL21 codon plus. Yes, there are literature on this and it should not be a difficult protein. I actually used 2hr induction but there was not much difference in banding pattern there either. I mean with overnight induction many more protein were there but i couldn't see the difference between induced and in-induced one except that only an 11kDa was in induced one, which was always there.

I will do western to see if it's the truncated protein or not.

I am also doubtful on my clone. Will re-sequence it to see what comes out.

The clone has been very difficult when re-culturing from the bacterial stock. I think i maybe have an instable clone. Then what i should do?

Best,

Mahbobeh

If you are sure of the C-terminus fusion of the His-tag it can not be truncated protein. If you find it in the not induced cells it maybe contaminant protein you are collecting in the nickel chromatography or an endoprotease activity on your recombinant protein sequence, check online for prediction

Do an expression

at 37oC for 4hrs with 0.1 mM IPTG. Process 1 mL culture to see the expression (Pellet down 1 ml culture, add 100 uL 1X sample buffer to boil for 5 min and load 10 microlitre on the . This is quick to analyze)

Did you also added "two" nucleotides between the last codon of your gene and the BamHI site in the reverse primer (for hexahistidine to be in frame).

Here is the possible causes-

1. Recheck your primers and sequence for frame shift mutation, this might encounter stop codon in between the sequences.

2. Have you biased your codon sequences according to E. coli, if not try Rosetta strain.

3. Try a western blot

4. Try a PCR (similar to colony PCR) from BL 21 cells with your specific primer, and check for the specific band.

5. And finally, it might be due to contamination; prepare a fresh competent BL 21 cells and try transformation once again.

Good luck..!