I want to produce and refold 32 mammalian amino acids that as a small protein in E.Coli. Has anyone got experience and suggestion which pET vector is suitable?
Have you tried it at all yet? You could just drop it into something like pET15b (will give you a His Tag) and see what happens. If that doesn't work, you may need a bigger tag, such as GST, NusA or thioredoxin.
We have used pET32a for expression of a 59 aa serine protease in E. coli BL21(DE3)pLys. It works well and we have got good results.
Not very difficult. we have cloned and produced a 13 aminoacid peptide by recombinant methods..you can attempt to express your protein as a fusion protein..with sites for chemical or enzymatic cleavage site to release the smaller fragment of your interest..good luck
sridhar
Thanks all just would you all let me know do i need his tag or any specific tag? Tanks in advance for your kind consideration
The benefits of adding a tag include (a) possible assistance in keeping the protein in solution and (b) gives you a handle on a rapid purification. You can always cut the tag off later - most vectors encode thrombin or TEV sites between the tag and the protein of interest.
As to which specific tag you want - it depends on what you have available, and what works for you. A His-tag is small, works well for purification and is usually easily cleaved, but often does little for solubility. I have used it with ~30 aa proteins with some success. Other tags may increase the solubility of the fusion (but you can still have issues if your protein is insoluble when you cleave it). You've really just got to try one (or 2, or 3...) and see what happens.
Small peptides are often flexible and thus protease sensitive. If you have problems with proteolysis of your peptide (tagged or not) consider expressing it as a fusion to an insoluble protein like KSI (Novagen sells vectors for this). You recover the protein in the insoluble fraction, then purify it under denaturing conditions. For these constructs, a methionine is usually placed between the tag and the sequence of interest. Cyanogen bromide can be used to cleave the construct at the Met residue. Alternatively, Asp-Pro sequences are acid-sensitive and have been used for specific cleavage as well.
This is exactly what I mentioned earlier (My post dated 25th May, 2012). However, we did not use the commercial vectors, instead, designed with whatever vectors that were available with us
May I know why the cloning of short peptide is such a difficult task?
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