I'm not sure what you mean by 'clone at the same side', but basically if one side it blunt and the other is sticky then just one fragment can be inserted. If the sequencing is ok then the cloning should be fine as well. Perhaps your protein is modified post-translationally by glycosylation/phosphorylation/lipidation etc. Or perhaps your protein forms di/multi-mers with itself or other proteins and its difficult to separate them. How do you treat your sample before loading for western blotting?

You get the vector sequence also when you are expressing inaddition to the cloned sequence. See the vector map and you get few additional aminoacids at the N-terminal end and some at the C-Terminal end. I cloned viral coat protein in pET 29 a vector which is 17.3kDa. I got additional sequence of 31 aminoacids anterior and 27 aminoacids posterior to the coat protein. So, an additional 58 aminoacids along with 17.3kDa protein. Each aminoacids give an extra molecular weight of 110Da . So, 58 aminoacids gives an additional 6.3kDa. Initially, i was searching for my expressed protein upon IPTG induction at 17.3kDa. Later, when i got this logic, i searched at 25kDa. Indeed, its expressing .So, you check accordingly.

Hi Sesha. I did that. Protein + V5 epitope + His tag should be around 68 kDa. However, the size of expressed protein is more than 100 kDa.

Amila, thats my argument too. since one side is sticky, the other is blunt, i dont think my insert got double clone in there. since its express in E. coli, so its not post modification. The expected size should be 68 kDa, but the expressed protein is more than 100 kDa. I thought is dimer too, but i heat the protein at 95 degree for SDS-PAGE and WB, so the protein should be denatured. Basically am out of reason.

I can suggest two explanations:

1. Your construct misses correct stop codon.

2. Everything is OK, but the apparent molecular mass of your protein is bigger than the real one. Did you do mass spectrometry?

I also agree with what Michael Agaphonov suggests. As a quick initial check, you might consider the following:

Does your protein contain a cysteine?

If so, do you have fresh reducing agent in your SDS-PAGE run or buffer?

If not, disulfide bonds can survive the preparation for SDS-PAGE and yield much higher (often 2x or greater) apparent molecular weights, as the disulfide bond(s) may be holding the structure together. 

Quick general reminder: DTT and BME both have fairly short effective lifetimes in solution, so always make sure they are fresh.

1) Proteins with pI less than 4 or higher than 10 often show apparent MW by SDS-PAGE 20-80% larger then calculated. You may wish to confirm MW of your isolate by gel-filtration.

2) Some proteins (especially containing Ig domain, but not only)  can form beta-swapped dimers (aka 3D domain swapped dimerization, see e.g. PMID: 8159715); such dimers cannot be separated by SDS-PAGE and may show apparent MW less than 2-fold larger than MW of a monomer.

3) If you used for you WB anti-tag antibody (anti-6His?), but not anti-your protein mAbs you can find falsely positive band. You can check it using the anti-protein mAbs. And for checking protein expression IP usually gives much reliable results than simple WB.

4) You may have "wobbling" stop codon - see e.g. PMID:11163755 

It may be fixed by cloning protein with 2 tandem stops.

You also can try either MS or measuring your protein functionality (activity or binding to specific ligand/receptor) etc.

But to save your time and efforts I'd recommend you just to make your construction anew and check expression by IP with anti-protein mAbs. Also you can try to express your protein in inclusion bodies - sometime it yields the best results.

Thank you all for your kind opinions. I will try to troubleshoot this problem