3 replies to this topic

[dhirendra]

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

[phage434]

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

[dhirendra]

phage434, on 14 March 2012 - 10:07 AM, said:

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

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...

[biolcrazy]

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.