1 reply to this topic

[ssray2000]

Hi, I am new to this forum and new to the area of protein overexpression in E.coli in general. I am trying to express a human protein in E.coli as a GST fusion using pGeX6P1. My general conditions for overexpression are to grow cells till O.D=0.5 and induce with 1mM IPTG and reduce temperature to 18C.
This method led to all the protein going into inclusion bodies   . So as the next step, we reduced the IPTG amount 0.05mM. This gave very little protein expression overall and it was impossible to say how much was soluble and how much was not.
  I read that there are E.coli strains that overexpress chaperones that have led to good refolding of problematic proteins. I was wondering if any of these strains are commercially available and if any of them work with pGeX6P1.
thanks to everyone in advance for their assistance

[T C]

Hi

Takara biosciences has a chaperone plasmid set pKJE8 I think (not sure about the name though).  We did order this plasmid set and used it.  The chaperones expressed very well but my protein was still in the pellet.  However, I visited a lab recently and they told me taht it worked very well for them.  You can give it a shot.

Best
TC

ssray2000, on Mar 4 2009, 07:13 AM, said:

Hi, I am new to this forum and new to the area of protein overexpression in E.coli in general. I am trying to express a human protein in E.coli as a GST fusion using pGeX6P1. My general conditions for overexpression are to grow cells till O.D=0.5 and induce with 1mM IPTG and reduce temperature to 18C.
This method led to all the protein going into inclusion bodies   . So as the next step, we reduced the IPTG amount 0.05mM. This gave very little protein expression overall and it was impossible to say how much was soluble and how much was not.
  I read that there are E.coli strains that overexpress chaperones that have led to good refolding of problematic proteins. I was wondering if any of these strains are commercially available and if any of them work with pGeX6P1.
thanks to everyone in advance for their assistance