Protein expression - (Jan/04/2010 )
I have a trouble in my expression expression work.
I am trying to express a tsetse fly gene in E.coli. So i cloned the gene (720bp) in pRSETA vector and confirmed it to be inframe and in proper orientation. This construct was transformed in BL21(DE3)plysS. I grew 5mls of the starter culture, inoculated 25mls of SOB (even tried LBmedia) and induced at 30deg, 37deg for 2hrs (even tried overnight): OD600nm of 0.6, 1.0, at different IPTG concs: 0.4-1mM. I can't really see on SDS-PAGE a clear band at the expected size in comparison with induced expression control (BL21(DE3)plysS transformed with pRSET vector), But there's a sharp decrease in OD600nm after induction indicating the toxicity of the protein!
I further did Western blot using HRP-His probe: No detection of the protein. I am thinking of growing a larger culture and try to purify it?????? Pls advice me on what to do.
Uhhhh!
bargul on Jan 5 2010, 12:30 AM said:
I am trying to express a tsetse fly gene in E.coli. So i cloned the gene (720bp) in pRSETA vector and confirmed it to be inframe and in proper orientation. This construct was transformed in BL21(DE3)plysS. I grew 5mls of the starter culture, inoculated 25mls of SOB (even tried LBmedia) and induced at 30deg, 37deg for 2hrs (even tried overnight): OD600nm of 0.6, 1.0, at different IPTG concs: 0.4-1mM. I can't really see on SDS-PAGE a clear band at the expected size in comparison with induced expression control (BL21(DE3)plysS transformed with pRSET vector), But there's a sharp decrease in OD600nm after induction indicating the toxicity of the protein!
I further did Western blot using HRP-His probe: No detection of the protein. I am thinking of growing a larger culture and try to purify it?????? Pls advice me on what to do.
Uhhhh!
i suggest u also try a lower induction temperature.....18 degree....
DRN on Jan 5 2010, 01:11 AM said:
bargul on Jan 5 2010, 12:30 AM said:
I am trying to express a tsetse fly gene in E.coli. So i cloned the gene (720bp) in pRSETA vector and confirmed it to be inframe and in proper orientation. This construct was transformed in BL21(DE3)plysS. I grew 5mls of the starter culture, inoculated 25mls of SOB (even tried LBmedia) and induced at 30deg, 37deg for 2hrs (even tried overnight): OD600nm of 0.6, 1.0, at different IPTG concs: 0.4-1mM. I can't really see on SDS-PAGE a clear band at the expected size in comparison with induced expression control (BL21(DE3)plysS transformed with pRSET vector), But there's a sharp decrease in OD600nm after induction indicating the toxicity of the protein!
I further did Western blot using HRP-His probe: No detection of the protein. I am thinking of growing a larger culture and try to purify it?????? Pls advice me on what to do.
Uhhhh!
i suggest u also try a lower induction temperature.....18 degree....
Thanks, i'll try that. I realize the sequence to have 39 E.coli rare codons! I'll try to use rosetta
I don't suppose you've checked to see whether your gene of interest contains codons that are rare in bacteria? I had a similar problem with an insect protein and had to use Rosetta 2 (DE3) cells from Novagen, which contain an additional plasmid, pRARE2, that encodes tRNAs that are prevalent in Eukaryotes but rare in bacteria. Good luck!
DRN on Jan 5 2010, 01:11 PM said:
bargul on Jan 5 2010, 12:30 AM said:
I am trying to express a tsetse fly gene in E.coli. So i cloned the gene (720bp) in pRSETA vector and confirmed it to be inframe and in proper orientation. This construct was transformed in BL21(DE3)plysS. I grew 5mls of the starter culture, inoculated 25mls of SOB (even tried LBmedia) and induced at 30deg, 37deg for 2hrs (even tried overnight): OD600nm of 0.6, 1.0, at different IPTG concs: 0.4-1mM. I can't really see on SDS-PAGE a clear band at the expected size in comparison with induced expression control (BL21(DE3)plysS transformed with pRSET vector), But there's a sharp decrease in OD600nm after induction indicating the toxicity of the protein!
I further did Western blot using HRP-His probe: No detection of the protein. I am thinking of growing a larger culture and try to purify it?????? Pls advice me on what to do.
Uhhhh!
i suggest u also try a lower induction temperature.....18 degree....
check the codon usage and preferrence in bacteria and organism you cloned it from. compare them some times the codon prefence differnce gives rise to lesser protein or even no protein production.