biovinz

make sure your Polymerase have terminal transferase activity.....otherwise you just gotto optimize the vector and insert ratio....sometimes purifying the PCR product helps....

SDS-Page check. İn Frame check.

You could try a cleavable Thioredoxin (Trx) tag. You just clone your gene in frame with the tag and you can cleave it off later. The Trx tag is supposed to be pretty good for increasing solubility of unstable proteins. We use pET32a-c vectors that contain Trx, S, His6 tags that are all cleavable.

how you know your protein is in inclusion bodies. did you add loading dye to bacterial pellet, boil it & load on PAGE?
you can use protocol for purification of protein from inclusion bodies.

You are right, the vector map available at ATCC website is not detailed.
But, you can have a look at this reference. This is what they have cited.
http://www.ncbi.nlm....pt=AbstractPlus

PhiX174 won't have native antibiotic resistance so presumably you are cloning some of it into a plasmid - how would you then select for the plasmid?

Note: I think this is a homework question, so I'm making you think a bit...

My overall conclusion to what you are describing here is that your protein is not soluble.

There are several possibilities:
1) what you see as good size protein after induction is in fact another protein that also is overexpressed when cells are induced e.g. a chaperone that is induced because your protein aggregates and the response of the cell is to produce chaperones not to have protein aggregates. In order to check for this I usually enrich my protein lysate in my protein by incubating 2 mL of cell lysate (after cell disruption and centrifugation) with 20-50 uL of glutathione beads; followed by 2-5 washes at lowest centrifugation speed. Like this you will not have to do a full purification before you know that you have only free GST. But be cautious: there are chaperones that bind to glutathione beads without having a GST -tag i.e. unspecific binding. But anyhow you would have the same chaperones present after the affinity purification step as well.
2) you do have your protein but it is found in inclusion bodies that are not in the cell lysate that you use for purification. This is why I always keep for an SDS-PAGE gel a sample of the pellet, clarified cell lysate and also this enriched fraction I mentioned above. Like this you know where you have your protein, if you have it at all. To be sure, I also do a WB against the GST tag (or the protein I am trying to express if I have a good antibody against it). Like this I would not confuse my protein with another protein that coincidentally has the same size.
3) your protein precipitates during the purification. This is why I do all my purifications in the cold room. Moreover, there is an experiment, called thermofluor through which you can screen several buffers in which your protein has increased stability. If you have such a buffer, your protein would not precipitate during purification. However, if you don't have this experiment established in your lab, it is a bit difficult to do. You basically need SYPRO ORANGE and a RT-PCR cycler for it, besides the commercial buffer screen (which is not at all expensive and you can use it for several years)

I cannot give you an advice about what to do to get your protein until you do some of the things above for us to see where exactly your protein disappears.

Andreea