I have cloned a gene for a 10 kDa bacterial protein in pET28a (to be expressed as a C terminal His tagged protein) and transformed into DH5a. I have confirmed the clone by restriction digestion and have sent for sequencing as well. Following fresh transformation into BL21 (DE3) cells and induction of cultures with IPTG (0.5 mM and 1mM) for 3-4 h at 37 degree as well as 24 degree celsius), I failed to see any over expressed protein on SDS-PAGE in pellet or supernatant fraction. Western blot of the same sample probed with anti-His antibody however showed a good band around 10 kDa; specially in the supernatant fraction; however the signal was similar in induced and uninduced control (absent in pET28a empty vector control). Can anyone provide an explanation and also suggest how to move forward with the purification.
Assuming you have correct gene in proper reading frame There are several things that can be done. simple things first-
media -LB, TB, auto induction.
cells - de3, star de3, rosetta de3(for rare codon)
construct - sometime putting the his tag at N terminus can increase the expression significantl. You can also try to express your protein with different tags. tags like MBP can greatly increase the expression.
codon optimization- you can try to get your gene synthesised with codon optimized for expression in E. Coli.
Biochemical techniques reasons
1) The western blot analysis is usually a sensible and specific method, so you have to optimize the proteins levels in the SDS gel and also the antibodies concentrations. Maybe you have a non specific signal with non induced control because of the great amount of proteins deposed on the SDS gel (more than 100 ng) and also a great concentration of specific antibodies. Sometimes researchers use the same amount of proteins (100 to 500 ng) for both dye coloration as Coomassie Brilliant Blue and the antibodies reaction In that case, you have to reduce the proteins quantity in the gel even between 10 to 25 ng and also dilute your specific antibodies (1 mg/ml) at 1/2500 or 1/5000 about 0.2 to 0.4 microgram/ml.2)
2) The recombinant protein could be expressed as Inclusion bodies so you have to optimize its extraction and solubilization of protein in the pellet by different testing agents asSDS, Urea, Glycerol, DMSO Reducing molecules (DTT, BME with especially anti-Proteases (PMSF)
3) Use Anti-proteases (PMSF, Trypsin inhibitor) for the soluble and supernatant extract.
Molecular cloning reasons
I agree with Dr Srivastava to verify the ORF orientation and also the Nonsens codon-stop in the cloned gene sequence especially in the recombinant vector of the recombinant bacteria.
1. Sequence the plasmid/insert and verify that everything is in order (promoter is there, etc) and 2 make sure BL21(DE3) cells do not destroy your plasmid.
I assume the clone and vector design are all correct. Some small proteins around 10k are hard to be expressed in E. Coli. system. I suggests that the protein is fused with other proteins such as GST or Fc et al. and then overexpress it. If you don't want the fused part, add a sequence between the tag and your protein, that can be digested by specific protease. Most of Anti-His antibody is not that specific and the positive bands you got may be just false positive.
Try the conditions used in this publication:
"Use of Rifampicin in T7 RNA Polymerase-Driven Expression of a Plant Enzyme: Rifampicin Improves Yield and Assembly." Alena Kuderová, et al., Protein Expression and Purification 16, 405– 409 (1999).
It has worked for me when I have had issues with inductions. Also you can try lower temperatures during the induction time.
For the expression of small peptides, we use Ubiquitin protein fusion. The fusion partner is removed by YUH1 and the target protein is protected from endoproteases during expression.
You can access the overview of this method on this link:
https://www.researchgate.net/publication/320877789_Biosynthesis_purification_and_chemical_labelling_of_AVPI_binding_motif_containing_SMAC_mimetics
Anything you need, contact us.
Good luck!
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