Hi Aglaia,

Based on my experience, I think there may be the following reasons:

1. There are some problems with cloning, for example, a stop codon appears in an incorrect position, resulting in early termination of expression. It is suggested to sequence the positive clones and then compares the sequencing results with the target gene sequence.

2. If the gene sequence is completely correct, or if there are only some synonymous mutations, you can consider whether it is a problem in the expression and purification process. Please check whether the appropriate concentration of protease inhibitor is added during the purification process or whether the purification temperature is low enough…

3. From the SDS-PAGE gel or gel filtration data, it can be judged whether the target protein is degraded during the expression and purification. If protein degradation occurs even when normalization of the operation is ensured, then it indicates the nature of the protein fragment itself is unstable. It is recommended to redesign the length of the construct or add a tag to avoid protein degradation.

Hope it helps!

Hi there,

So you used to express the entire protein successfully and now you get a smaller product. 2 obvious possibilities: for some reasons the product is now sensitive to partial degradation during the expression/extraction process (are the conditions exactly the same from the early experiment to the recent ones?), the clone you use as the starting material has evolved/derived. The BL21 background is known as unstable and susceptible to recombination leading to genetic modifications. It is advised to perform transformation with the expression plasmid (which is stable in vitro) everytime you intend to perform expression experiment.

Thank you all very much! It turns out the clone I used had probably undergone some mutation. I performed new transformation with the expression plasmid and the overexpression turned out fine

Great to hear. Glad you solved it. If all your cloning was good, the problem had to be the colony or procedural in the purification. Fresh transformation is always good.

in addition to all mentioned above, please be noted that, in SDS protein can appear shorter than its expected size! Check the sequence of your clone and do WB with specific antibody.

it is not advisable to use a colony from the same plate. The better thing to do is to make a glycerol stock of a colony that gave you expression of the correct version of your protein (90 kDa). You never know what went wrong with the new colony you picked.

Start a new transformation with your plasmid and Pick a new colony. If you get expression of the correct protein, then do a glycerol stock.

@Aglaia, a late comment, but I would suggest to kept your strain under repressed condition until you need it to be expressed. Under unfavorable production of a protein that plays no role in fitness, a mutant with deactivated production can taken predominance in the population very quickly.