How do you check for overexpression? What kind of protein do you want to overexpress? Some of them (membrane proteins for example) are hard to overexpress in a soluble form.

Try to express the recombinant protein in BL21 (DE3) strain.

CMV mamalian vector will not work in Bacteria. Also Dh5 alpha is not used for expression of proteins as it doen not have T7 RNA polymerase engineered in it. You clone your intron free gene with methionine at the N-terminus in any of the pET vector under the control of T7 promoter. Transform the vector into E.coli BL21(DE3) strain. Grow the resulting transformants in LB medium till it reaches an OD of 0.80. Add 1mM IPTG and continue incubation for another 4 hours. check the protein expression on SDS PAGE.

Completely agree with Nagaraj answer, check what is the promoter on your CMV vector, to see if it possible to induce with iptg, and also it need to have a replication site compatible with bacteria

Thank you very much all for your valuable suggestions. The vector is pFLAG-CMV1 vector and the gene of interest is a membrane protein. The promoter is CMV promoter. I have tried to induce with IPTG from 1mM to 4mM for 4 hrs, but still didnt got any expression. I will now then try to introduce the vector in BL21 strain and then express it using IPTG.

for membrane protein you should use strain like C41(DE3) or C43(DE3). but i am not sure if your promoter is induced with iptg. i think you need to clone it into another vector

Dipon,

I believe I understand your problem and the interpretation of the answers given. The pFLAG-CMV-1 vector indeed has the T7 promoter and lac operon. However, the problem is that the orientation of the T7 promoter opposes the CMV promoter. Any transcripts made from the bacterial promoter, therefore, will be *antisense* of your gene of choice. The existence of these bacterial expression sequences comes from the fact that the parent vector of pFLAG-CMV1 was probably a derivative of pUC18, which can indeed be used for bacterial protein expression in strains such as BL21.

I totally agree with Nagaraj and Sofia. You definitely need to go back to cloning. Any attempts to express your protein in vector you have now will be futile as the most you will get is a 8kDa protein of rubbish coming from sequence mostly of the polyA sequence.

How you go about the cloning will depend on how you wish to use the vector later on. If you also wish to use the construct for mammalian work and bacterial expression, then you can use the pDUAL vector from Stratagene. It has both the CMV promoter as well as the T7 promoter with the lac operon. If you want to keep your FLAG-tag, you can PCR out a fragment from your current construct and add the Eam1104 I restriction sites via primers. The manual for the vector is attached.

The next question is why you wish to express the protein in bacteria. If you are planning to use the product for antibodies, you will ok (see below). If you are doing functional assays, you will most likely have to find another expression system such as yeast or insect cell culture (I am assuming that you are interested in a mammalian membrane protein). Bacteria cannot do the post-translational modifications needed to process mammalian membrane proteins properly.

If your intent is to produce loads of protein for antibody production, bear in mind that you will be most likely dealing with proteins in inclusion bodies as membrane proteins tend to have hydrophobic residues and the bacteria will not be able to process them properly for lack of organelles and internal membranes. You may have to purify your protein by more traditional biochemical means (e.g. denaturation by urea, ammonium sulphate cuts, reverse phase chromatography) rather than by the easy means of a FLAG epitope as it may not be efficiently exposed to solvent. Furthermore, FLAG-purification depends on anti-FLAG antibodies interacting with the tag, so any "purification" in a chaotropic agent such as urea or guanidine-HCl will result in nothing.

A better strategy if you are intending to do what I described above would be to use a 6xHis tag fusion protein expressed from an appropriate vector. If you have inclusion bodies, then you can denature your extract with urea and pass it through a nickel column. Take care about the pH of your buffers as binding to a metal column depends on your imidazole groups to be deprotonated. Drops in pH will decrease your protein's binding efficiency. Note that buffers change pH with temperature, so a purification may work in the cold room may not work at room temperature.

His-purifications are known to be dirty (high background of contaminants) as there are a lot of proteins in the cell that have histidine. It is best to add a bit of imidazole (10mM final concentration) to your extract to prevent non-specific binding [don't forget to *omit* EDTA or any chelator from buffers!!!]. After extensive washing with 10mM Imidazole buffer (>10 column volumes), elute using a very shallow gradient. If you need a protocol, message me, and I will be happy to explain more.

Best of luck!

http://www.genomics.agilent.com/files/Manual/214501.pdf

I am using pET & pGEX bacterial expression systems. They works fine in my lab. If you want to overexpress yr protein in E. coli bacterial system, please contact me at [email protected]

Shaharum