What is best method for enhancing the expression of a gene if you need a protein, is it increasing copy no or removing control from it or anything else?
Hi Nadia,
Do you mean you want to study the protein, and therefore you want to produce a lot of it?
In that case, the best method is to clone the coding sequence (cDNA) in an expression vector, where it will be under the control of a strong promoter and other regulatory sequences. By transfecting a suitable host cell, you can then produce large amounts of your protein.
For human genes/proteins, a gene can for instance be cloned in the pBABE vector and this can then be used for lenti-viral transfection into 293T cells.
See here for some more information: https://www.addgene.org/empty-backbones/
Agree with the above comment ... also, to maximise translation of an overexpressed cDNA sequence it is critical to use a strong kozak consensus sequence surrounding the ATG (encoding the N-terminal methionine) ... i.e. accATGg configuration is most optimal, if the second amino acid codon will allow. These base changes are simply engineered into the expression vector by PCR, or site directed mutagenesis. For maximal protein expression, I would also recommend using 2 tandem stop codons i.e. TGATGA at the 3' end of the open reading frame. Finally, in addition to a strong viral promoter (as suggested above) it is equally important to include a strong transcriptional termination sequence at the 3' end of your cDNA. As an optional extra, forcing an mRNA splicing event by introducing a synthetic intron is another means to further enhance protein expression levels.
In addition to the above be aware also that if expressed to a high level some proteins have a propensity to drop out of solution and form inclusion bodies which lowers your yield and creates a host of downstrem issues. Placing the the protein under control of a strong inducable promoter might be advisable.
Hello Nadia.
1) Have a look at the link with a similar topic. https://www.researchgate.net/post/How_to_clone_a_gene_from_total_RNA_to_overexpress_it_in_cells
2) Decide cDNA to be cloned, amplify in a flask culture to obtain large amount of plasmids. You need cleanest DNA for maximum overexpression. This is the key for successful overexpression. Therefore, make aliquot tubes of DNA for separate cleaning and re-precipitation to get real clean DNA molecules.
3) In addition to the above helps, I suggest a few tips for general purpose of overexpression to make work load less to get enhanced overexpression. You coud use a simple vector optimized for strong expression vector, pCAG promoter. You can obtain from Addgene (pCAG-GFP).
4) Use Lipofectamine, so efficient to get transfectants with high copy numbers of your gene.
5) Maintain your cell culture clean as much as possible. Use active cells with ~30% confluent cells. Make sure all solutions and buffers involved clean.
6) Finally, use 5 times higher DNA concentration of the recommended. Work fast when you use Lipofectamine.
Best wishes.
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