I have a protein of 277 aminoacids. I want express a truncated version of that without first 22 aminoacids from N-terminal and also the sequence should start with a cysteine residue. Is there any method to do this?
See this link for more information about the SUMO protease necessary to remove your N terminal SUMO fusion partner (leaving a Cys as your first residue if you clone it that way)
http://tools.lifetechnologies.com/content/sfs/manuals/sumo_protease_man.pdf
If you are using E. coli as the host, prepare a SUMO fusion. The only residue that does not work in the P1' position is proline, if memory serves well.
See this link for more information about the SUMO protease necessary to remove your N terminal SUMO fusion partner (leaving a Cys as your first residue if you clone it that way)
http://tools.lifetechnologies.com/content/sfs/manuals/sumo_protease_man.pdf
A very comfortable way is the expression of the target protein fused to an intein (https://www.neb.com/products/e6901-impact-kit), which at the same time offers a convenient and efficient purification procedure (chitin binding domain)
We have expressed a protein with a cysteine following the first Met in E.coli. Then we found that Met is cleaved in E.coli by Mass spectroscopy. See Kao et al. Bioconjugate Chemistry 2008.19, 1124-1126. When the residue after the first Met is a low MW residue, Met will be cleaved. It is true for my another protein which has sequence "MG... ". So you could probably just directly try to express your protein with a Met in front of your Cys in E.coli.
@Rita,
I had always assumed that N-terminal Met removal would be incomplete when Cys was the second residue (per PMC298257). Was your setup sensitive enough to detect minor species? (Sorry for not looking at your ref, but it was behind a paywall)
You may introduce an enterokinase cleavage site (DDDDK) followed by cysteine as your first aminoacid residue. Enterokinease cuts after the lysine, leaving no extra aminoacids in the product. The only requirement for efficient cut is that lysine should not be followed by proline.
In addition, the DDDDK motif can be used for detection in a Western blot or for affinity chromatography.
@Alejandro
According to our mass analysis, it is cleaved. Not minor species. Of course I could not guarantee for other proteins. Anyway, it is worth to give a try.
Look at Haas, Orevi; Biochemistry (2014?). They wanted exactly the same, a cysteine at position zero. But to be able to obtain the protein by recombinant expression they had to put methionine at "position minus 2". You always have to start with Met in that case. The only other possibility is chemical synthesis which is as of now out of the question if the target sequence is longer than 30 residues. Best, success, Maik
Express the protein with an enterokinase cleavage site followed by a cysteine residue and once cleavage happen you should have a protein with a cysteine at its N-terminus.
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