Bpaul

Your protein is not soluble in the conditions you are trying. When a GST tagged protein leads to the 25 kDa band and not full protein, that means that the cell does not like the protein (either toxic or insoluble, or not properly folded) and it is cleaving it. GST is not a tag that improves folding. It is a tag tag gives false solubility. Better use a MBP tag or a NusA tag. Here a paper talking about this:
http://www.ncbi.nlm....pubmed/16168669 (let me know on private if you cannot access the article, I can send it to you)

You have already tried different expression conditions in this vector. People usually start with 3 different tags in 3 different vectors with different promoters of different strengths (hence they clone in parallel 9 constructs) to try them:
http://www.ncbi.nlm....pubmed/20814932
http://www.ncbi.nlm....pubmed/18678258 (this one has a nice scheme on the second page about what I mean above)
You can also try chaperones system (co-expressing your protein with different chaperones)
http://www.takarabio.../pdf/hd/BV1.pdf
Or in ArcticExpress:
http://www.genomics....tail&PageID=467
since chaperones do not function at low temperatures, in these cells people have cloned some chaperones from sychrophilic bacterium Oleispira antarctica. This makes it possible to both use chaperones and low temperatures.
Another thing you can try with the construct you have now is the autoinduction media, sometimes in this the protein is folded better:
http://www.ncbi.nlm....pubmed/15915565

There are many things you can try. There is no direct way than trying. The order is up to you. Good luck,

Andreea

Sequencing machines are not usually able to read the first 10 to 20 nt of your sample. This is from both sides, 3' or 5'. That is why most people do not use the PCR primers for their sequencing. Instead, you can use the primer of your plasmid's promoter.  Every expression plasmid has a promoter right before the MCS site. If your PCR product is not long (less than 1000 bp) you must sequence with the primer of that promoter (usually T7, T3 or CMV), if it is longer than that you can use multiple primers. You can design primers that bind to the regions inside the PCR product and ask the company to sequence your plasmid with those.

Moreover, when you send sample for sequencing you need to always talk about DNA sequence, strand, 3' and 5'. Using amino acid or protein sequencing in such conversations are not professional.

I can add that we do not send ligation reactions for sequencing: we first transform the ligation into a cloning strain bacteria i.e. DH5alpha, XL1Blue... then miniprep the plasmid and send that for sequencing. What use is to PCR the ligation and then send it for sequencing? Of course you will PCR your insert, ligated or not. It is a waste of sequencing labels (7 € per sequencing reaction) to sequence something that you are not sure you can amplify as a plasmid. What would happen when you transform it and it did not work? Or it is not ligated at all?

Andreea