My pET28 clone containing 3 flag-Avitag and TNF alpha fusion protein is not expressing in E.coli Bl21 (DE3) cells. Sequencing results of the clone showed that all the sequences are in frame.
As you know, there is no guarantee that all proteins can be expressed nicely in E.coli; Solubility is the big issue for making recombinant protein in E.coli. Check cell lysates and pellet, with a western blot. Perhaps your protein can be found in insoluble fraction which tells TNF (human origin?) is insoluble in E.coli.
Is your construct optimized for expression in E.coli? Sometimes, modified tRNAs are required to express certain mammalian proteins in bacteria. If your construct is not E.coli-optimized, the bacterial cells may be encountering problems with translation.
Also, I second Ananda's comment. Solubility is to be checked.
Best,
Sagar
1). I will be used different concentration of iPTG (if this plasmid have iPTG inducible promoter).
2) I will be run protein gel with total lysate of induced (in different conditions and concentrations) and not induced cells and do western
3) Sometime protein especially from foreigner host (not E. coli) can be toxic. In that case you can look for different expression system. In that case purify culture after induction and sequence plasmid from 3 independent clones.
4) If you are purifying protein – remember about solubility. If you do not deed native conformation you can purify under denaturation conditions.
If you do not see anything in step 2- most likely E.coli do not like you protein
Did you check your sequence for rare codons? http://nihserver.mbi.ucla.edu/RACC/
The sequence I want to express has rare codons so I decided to express in BL21-CodonPlus(DE3)-RIPL strain that contain extra copies of the argU, ileY, and leuW tRNA genes.
Thanks all for your valuable suggestions.
I have induced the expression using 1mM IPTG and at different temperatures i.e.37,28 and 15C. I have checked the soluble and insoluble fractions by coommassie as well as Western blotting.
In the same vector, I have expressed the His-TNF which was very well expressed.
If you got expression with a similar construct, your sequence is fine and you dont have any product (no insoluble fraction???), I would start over with transformation of the sequenced plasmid into fresh BL21(DE3). Maybe even consider to sequence after transformation into BL21.
If formation of inclusion bodies is your actual problem, go for overnight expression at reduced temperature (15 to 20C) and reduced inducer concentration (1mM IPTG is in my opinion to much for this expression system, please try 0.1 to 0.2 mM). I could get more soluble protein by adding 0.5 M D-sorbitol(final concentration) to the medium (there are some publication around, sorry for not ading a proper ref.)
Good luck!
I'd do a miniprep and restriction digest to check that the plasmid is still intact. If your plasmid makes E. coli sick, deletions may be selected. If this is the case, you will probably have noticed variable colony size on plates the first time you streaked out the clone.
The bacterial strain you are using is not optimized for human codon usage and you may witness truncated products of your desired protein.Try using BL21(DE3)pLysS strain which contains the genes for the rare tRNAs that are required for expression of any human protein in E.coli.This strain would need chloramphenicol(34microgram/ml) along with your selection antibiotic(amp for your pET system) for the maintenance of the plasmid for the tRNAs.
I am using the strain which have tRNA plasmid, in pet 53 but still I am not getting protein......
can you suggest.......
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