desired protein is going in pellet...do not want to go for renaturation - (Mar/14/2012 )

I am working on a gene which seems to cause some sort of toxicity (observed as per the no. of transformed colonies in the plate).
I have cloned it in pGEX-6p1 and pET 28a. and expressed it but the expressed protein afer sonication when ran on SDS-PAGE is going in the pellet. so it seems that my protein is forming inclusion bodies. further I want to crystallize the protein. so I do not want to compromise with the concentration and structure of the protein. Please suggest me some cheap methods. ( the size of gene of interst is 966bp).


Thanks,
dhirendra

Induce less aggressively to lower the amount of protein being made. Culture your cells at lower temperature (25-30C, possibly lower).

phage434 on Wed Mar 14 18:07:24 2012 said:

thanks for the reply. but i already did that.. i have decreased the induction time and IPTG concentration. cloned and expressed in both dh5 alpha and bl21 cells.. with 16 degree as my induction temp...

This might sound weird but you can try this out. I am expressing my protein from pGEX6p1. Initially when I grew the cells and induced with 1mM IPTG at 37C, my protein was present in low concentration but I could get it in the soluble fraction. But this week, I decided to induce the bacteria at lower temperature (25-30 C) to increase the yield of protein and guess what... all my protein is now in the insoluble fraction! The only explanation I can think of is that if the protein is expressed above a particular threshold, the bacteria seem to put it in the inclusion bodies.... Try doing the induction at 37C.