I have to clone and overexpress a gene, upon analysis of the encoded protein sequence with Expasy ProtParam tool, it is showing a PII of >40, saying that the protein is unstable. How significant is the PII value?
Hi
I express a chimeric in silico designed protein with high instaility index in my PhD thesis. I think this index is not very significant if you express intrested protein as fusion with GST or Thr tag. As alternative you can express your protein as inclusion bodies (expression in 37C).
these strategies is applicable.
Be succesfull.
I also had to express an incredibly unstable protein and it was certainly much less problematic when fused to a GST-tag. However you can try using Sarkosyl, a detergent, to purify from inclusion bodies, see this ref:
TAO, H., LIU, W., SIMMONS, B. N., HARRIS, H. K., COX, T. C. & MASSIAH, M. A. 2010. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-‐100, and CHAPS. BioTechniques, 48, 61-‐4.
It may be that your protein expresses just fine - give it a try - but if you see it is very insoluble then Sarkosyl or the GST-tag might be the way. However you may not want your protein tagged - and when I cleaved off the GST-tag I found that the protein wasn't detectable any more on an SDS-PAGE gel (although we could detect it by NMR).
Good luck!
High protein instability index depicts loose bond angles or any pH, temperature, heat sensitive protein structure. In solution such proteins can be obtained by changing most interactive and backbone bind angles in total, side chains or sheet instability shows a possible three D change.The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein are also found to influence its solubility on overexpression. There are thermostability proteins contain amino acids which have, in vivo half-life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli.
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