fail on express recombinant protein

[happy725]

I have problem with expression of my recombinant protein, which will be used to raise Ab. I cloned a full gene (954bp) with a 6his tag at 3' in frame into 2587bp pRUN (modified from pET)expression vector using C43(DE3). DNA sequence was confirmed. A O/N culture was refreshed until OD 0.4, and IPTG added to finial 1mM (~4h). 1ml culture were tested before and after induction, SDS-PAGE showed the extract same patterns. The gene has 83%GC rich exon 1 sequence (83bp), is that could be a problem??

[happy725]

I have problem to express my recombinant protein, which will be used to raise Ab. I cloned a full ovine gene (954bp) with a 6his tag at 3' in frame into 2587bp pRUN (modified from pET)expression vector using C43(DE3) strain. DNA sequence was confirmed. A O/N culture was refreshed until OD 0.4, and IPTG added to finial 1mM (~4h). 1ml culture were tested before and after induction, SDS-PAGE showed the extract same patterns. The gene has 83%GC rich exon 1 sequence (83bp), is that could be a problem?? The protein has unknown function, and predict to be a toxic protein. What shall I do?? Thanks

[danfive]

Two things come to mind:

1. Make sure you are comparing exactly Xug of pre-incubation lysate to Xug of post-incubation lysate. This is easy thus first, really not that important, IMO.

2. Just perform either western blot or Immunopurification for His-tag protein. This is the critical part to let you know you have expression.

Don't worry so much about expression at this point, E.coli will express anything. What is important I think is the size of the recombinant 954bp equals how many amino acids?

any way go after the His tag.

[happy725]

Thanks danfive for the suggestions.

I will perform either western blot for His-tag protein. The size of my recombinant protein is 312aa, would this be a problem for expression??