I am trying to express and purify a viral cysteine-protease inserted in pET28a expression vector. The host is E.Coli BL-21 (DE3). I have done the transformation experiment two times and colony was formed beautifully in both cases. I have used IPTG for induction in a concentration of 0.2mM, 0.5mM, 1.0mM and 2.0mM, each for 4 hours, 5 hours and overnight (14 hours); both at 37 degree and 18 degree. During induction the bacterial OD was 0.7 to 0.9 in each cases. The problem is, I have not seen the desired band in any SDS-PAGE gel. There is no sign of IPTG induced expression. In fact, there is no band at all at the desired place. I have tried purifying the protein in hope of finding at-least something, but, the OD of the eluted fractions with five different imidazole concentrations (100mM, 200mM, 300mM, 400mM, 500mM) did not give away any peak notifying the presence of the purified protein. Please give me some advice regarding this problem.
The target protein is not toxic as the bacteria are alive and kicking and bacterial proteins are being expressed with no difficulty. The sequence is not near the start codon of CDS either.
Dear Banadiba
expression of proteases and expecial viral or mammalian enzyme in E.coli could be challenging in E.coli for several reasons as:
- the protease activity may result toxic for the bacteria and lead to formation of IB
- the dna sequence may contain several rare codons which may prevent or drastically reduce protein expression:
In this case:
1)Did you check for rare codon?
You can check it at
https://people.mbi.ucla.edu/sumchan/caltor.html
If you protein contain many rare codons you can try to :
- Express it in Rosetta (DE3) strain
- Order and clone a synthetic gene with codon optimized sequence
- Add a N-terminal of your clone a fusion tag as GST, MBP, GB1, trx that may enhance protein expression
You can find a summary of all this possible tools in the presentation available at the following link
https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG
- the protein may contain structural S-S bonds that are difficult to be formed in the E:coli.
In this case you can try to use the Origami (DE3) E.coli strain or add a pelB or ompA leader at the N-terminus which can allow periplasmic expression in E:coli.
- The protein may contain leader peptide usefull fo the protein secretion in vivo but which will not work in E.coli and have to be removed.
You can check for presence of leader peptide at
http://www.cbs.dtu.dk/services/SignalP/
Do you already check all the previous point?
best regards
Manuele
Thank you Manuele for your precious input. I will check for these problems immediately
- Badges
- Science method