We have purchased an expression vector from DNASU (pANT7_cGST). The expression vector has a human protein cDNA as insert. we have made multiple attempts to express the protein in different con of IPTG from 0.05mM to 2mM and also at different temperatures from 16 degree C to 37C. But still we have failed to express the protein.
I've got a few questions:
-Do you check, whether your bacteria have the correct plasmid after transformation?
-Is the protein (its DNA sequence) you want to express in-frame?
-In which medium do you grow your bacteria?
-What protein are you trying to express?
Just a few ideas:
-I don't know your vector, but maybe another one could help… In my case I used pET-vectors (25 & 28) and it usually worked fine.
-I had better bacteria growth and protein expression when I did autoinduction (see the paper below).
Studier, F. W. Stable Expression Clones and Auto-Induction for Protein Production in E. coli. Structural Genomics: General Applications, Methods in Molecular Biology, Chapter 2 1091, 17–32 (2014).
Adrian Marc Hauri , we have purchased the clones from DNASU and we have been told the clones are inframe and we have confirmed correct plasmid insertion after transformation by PCR and restriction digestion. We use Luria berttani broth for protein expression. Even pET vectors failed to give us any protein expression.
Strange...
Are all the necessities present in your plasmid(s) (e.g. promoter for efficient transcription / ribosome binding site)?
Did you also sequence your insert/plasmid? I used to always sequence my "correct" plasmids to be a 100% sure.
Is your protein a membrane protein? You'll surely have troubles with the amounts and with extracting it then...
Hi Vinay,
As Adrian Marc Hauri pointed, first sequence the construct and make sure that the insert is in correct frame. This also gives you the understanding if there are any unwanted mutations.
As the cloned gene is derived from cDNA, there is a high chance for the existence of rare codons in the cistron. Hence, try to express this construct in Rosetta (DE3) strains that encompass pRARE plasmid.
In addition, as a control, you can also check and confirm if the stock of BL21 (DE3) and IPTG are fine by expressing some other working plasmid from your lab .
Dear Vinay,
Years back I had purchased the same vector with human gene insert and tried expressing in BL21 DE3. I tried all the possible IPTG concentrations, temperature variations and other optimization steps. I did see induction of the protein but at a higher molecular weight as expected and upon purification, i did not get any protein. You can try the following-
1) Check if you also see induction at an aberrant molecular weight.
2) Try Rosetta (DE3) strains for induction and purification.
I would highly recommend you to sub-clone your gene of interest in either PGEX (For GST tag) or pET ( For His Tag), as these vectors have been thoroughly used for protein work and troubleshooting will be easier.
Also, I would like you check both supernatant and pellet fraction. Did you see any expression in any of the fractions?
Good Luck
Thank you all will look into the suggestions and attempt to implement it.
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