I am in desperate need of advice.
I did transformation of BL21 (DE3) pLySs, Rosetta 2, and T7 Express with my genes of interest cloned in pET30 a vector. I have a recombinant e coli protein in pET30a which showed a no protein expression level. I cannot see the band on SDS gel. I tried different competent cells like BL21-CP and BL21-DE3 and got same results. I tried different temperature for bacterial culture and got the same results, I changed the IPTG concentration 1mM and 0.5mM, no change. Original sequence is confirmed by sequencing. If you have any idea this would be great .
Yes there is such a relationship -- under normal (non-extreme) conditions, the limiting factor is the translation initiation rate. So on average, other things being equal (which btw they're not), you'd get a fixed number of molecules. That means, shorter proteins make less mass, and less band intensity. That said, you can certainly express many small proteins to detectable levels. That's not by itself the problem.
More likely, your problem is that E. coli simply doesn't like your particular protein. For proteins from different kingdoms, this is common. There isn't usually a reliable cure for that within the confines of the native sequence. You'll need to use an N-terminal fusion tag, which initiates frequently in E. coli. Such as GST, MBP, HQ, GFP. You can use that to transfer the tag's high initiation frequency on your protein.
Potentially, you'll need to cleave the tag off later, with a protease such as TEV. This is cumbersome and annoying. But it's the only reliable way to do it, if E. coli doesn't like your sequence.
Hi,
If you have insert in vector confirmed by sequencing, your protein may be expressing. If you have His tag try to purify it and do Silver staining to detect. It can sense protein as low as 1ng on SDS gel.
Good luck
Just in case,
I imagine that you see the protein expression by SDS PAGE, are you sure that are not loosing the band of your protein? Because is to small and run faster than the comasie of the marker
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