modified pET30b

[JohnDakota]

Hi Everyone,

I have a gene of interest that has an important membrane binding domain at it's N-terminus. It was already designed with an 5' NcoI site, so I was just going to use the NcoI site in pET30b however there is a fairly large N-terminal affinity tag in PET30b. What I've done is cut the N-terminal tag coding sequence out with NdeI and KpnI, treated this with Klenow to blunt, and religated to generate what is essentially a pET30b vector lacking the N-terminal tag, and will now have the ATG start of the NcoI site.

This vector was confirmed through dna sequencing.

I was wondering if anyone done this before and it's worked, or if pET30b requires the NdeI site to properly express recombinant proteins. I have been having problems expressing protein in this modified vector. I should mention I haven't tried expression in the original pET30b yet simply because I had this modified vector on hand.

[Andriy]

Hi John

You strategy for removing NdeI - KpnI fragment seems sound, as long as you have your T7 promoter the expression should work fine

At the same time, there are just so many proteins that don't express well in E. coli. And so many factors that people adjust to change expression levels - time of induction, IPTG amount, growth temperature, E. coli strains...

Also, you mentioned that you have a protein with a membrane domain - that should be a tough one

So, assume that your construct is OK and look into other factors

Andriy

[Andriy]

Hi John

You strategy for removing NdeI - KpnI fragment seems sound, as long as you have your T7 promoter the expression should work fine

At the same time, there are just so many proteins that don't express well in E. coli. And so many factors that people adjust to change expression levels - time of induction, IPTG amount, growth temperature, E. coli strains...

Also, you mentioned that you have a protein with a membrane domain - that should be a tough one

So, assume that your construct is OK and look into other factors

Andriy