1. Did you use PCR and sequence to check the  protein-pET28?

2. Is your protein soluble or not ?

3. If you did not find the band, how to  varified the expression conditons?

Thank you, Hyde!

The expression at lower temperature (26 oC) gave soluble protein(s) but the lane on the GEL was almost a protein ladder. As you recommended, I will resort to sequencing the plasmid as the gene was already sub-cloned into the vector during the purchase.

What is the origin of the gene you are trying to express, eukaryotic or prokaryotic? If its eukaryotic you will need to check the DNA sequence to check the sequence for rare codons i.e. codons that E. coli doesn't normally use Arg, Pro, Ile, Gly, Leu. These rare codons i.e. E.coli has very little of these tRNAs and will therefore not be able to express the gene. It is especially difficult when one of these codons are located in close to the start of the gene as they will cause the RNA polymerase to "fall off" and thererfore no transcript will be made. If this is indeed the case you can switch to a yeast expression system like Pichia pastoris. One advantage of this would be that  yeast secrete their protein into the growth medium so no lysing of cells etc.

If you determine that E. coli wont have expression issues then set up an experiment to compare soluble vs insoluble fractions. Start your experiment by inoculating an overnight culture with a single colony from a fresh streak plate on Kanamycin (make sure your cells contain the plasmid/gene by doing a colony PCR to confirm the presence of your gene in the transformed cells). The next day inoculate your media (containing no antibiotic). Before you induce expression take a 15 ml aliquot at time 0. Spin down cells and resuspend in 1 ml protein extraction buffer and freeze at -80. Induce expression at OD = 0.4 using 1mM IPTG and take 15 ml aliquots every hour for 4 hours. As before spin down the cells and resuspend in 1 ml of your protein extraction buffer and freeze at -80. The next morning start your protein extraction by thawing your cells. Once thawed divide the cell suspension and place in 2ml eppi's and place on ice. Set up the sonicator. Sonicate each cell suspension for 10 sec burst and resting on ice for 10 sec. Repeat this for each aliquot 4 times. Once sonication is done centrifuge at 10000 rpm for 10 min at 4 deg. to separate insoluble and soluble fractions. Remove soluble fraction and place in fresh eppi's. Resuspend the insoluble fraction ( i.e. cell debris) in 500 ul protein extraction buffer. Perform a Bradford assay on all the fractions. Run a SDS-Page gel of the insoluble fraction on one gel and the soluble fraction on another. Load 50 ug of protein per lane in sequence i.e. 0 hrs, 1 hr, 2 hrs etc.

What you should expect is that their should be an increase in the thickness of the band over time in the size range you are expecting (determine this beforehand so that you know how big your protein is going to be and where to excpect it). So, time 0 should have no band and 1 hr to 4 hrs should see an increase in band width. The reason for the second gel, is to see if the band you seek is in the insoluble fraction. Instead of speculating you can be sure. If this is the case you can either go the yeast expression route or you can try and solubilize using uea etc.

Hope this helps

Cheers

I would suggest to express a reportergene (RFP or GFP) which was cloned in the same vector in a parallel expression experiment - if the cells turn their color to red or green, then you know that you have no technical problem in your experimental setup - keep in mind that not all proteins can be expressed solubly in E. coli.

I forgot add one thing. Do you know if your gene is inframe with the promoter? Have you confirmed this with sequencing? You might think its inframe simply because you cloned, but this needs to be done by sequencing and subsequent in silico translation. The promoter, his-tag and gene should all be in one open reading frame when analysed.