I want to clone a 1290 aa long bacterial secretory protein having signal sequence (33 aa), autotransporter domain (at C terminal) and hydrophobic domain (acts for main function of protein) and disordered regions. I have pET15b, pET28 and pET30a expression vectors and E. coli DH5alpha & BL21. Should I clone whole protein or in segments, it has three globular domain starting from 1-318, 426-827, 872-1290. I'm also wondering whether I should consider signal and autotransporter domain for cloning or not. What another parameters I should consider to this protein.
Agree with Sebastian in both difficulty of hte task and the possibility of prepare something shorter (evena peptide) to answer your specific (scientific) question.
An intrinsically disordered region will make your protein very susceptible to proteolysis, both inside the bug and during purification. Depending on what you need the protein for, it may be possible to just leave it out, that would simplify things greatly. Of course, if that region is responsible for the protein-protein interaction you want to study, then you'd just have to bite into the sour apple.
Your hydrophobic domain, is it by any chance trans-membrane? That also would complicate things.
What about posttranslational modifications, especially glycosylation? Bacterial expression of glycosylated proteins is a known bitch, you may have to switch to yeast or even insect cells.
Hi Sandeep,
You can start this project by fragmenting your protein into 3 parts and start GST-tagged cloning of the fragments. Further you can check using the Binding assay protocol. The parts are to be confirmed via western blots. Thereafter, you can further fragment the confirmed clone and repeat. By this methos you can reach to the amino level interacting region. Further, confirm it in-vitro using mutation studies.
Good Luck.
Pratibha
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