Hi Gerry, In one case, I had a mutation in the ribosome binding site sequence. If you haven't already checked, it might be worth checking whether the 5'- and 3'- junctions close adjacent to the ORF are all correct to make sure the transcriptional and translational elements do not somehow contain mutations.
On occasion, figuring out what is wrong with the current construct might send one on a endless wild goose chase. So if a bunch of ideas with the current construct fail and you still want to try and express in the pET system before moving on to another system, I recommend the pET Expresso system. No need to ligate etc. Add clean PCR product + vector + cells and you directly screen for clones that are made by homologous recombination in bacteria. Cheers, Raji On Sat, Mar 17, 2012 at 2:26 AM, Jerry McCully < [email protected]> wrote: > Dear ALL; > > Recently, one of my colleagues cloned a gene (200aa) into pET30a > vectors with either a N-ter or C-ter His6 tag. The correct reading frame > was confirmed by sequencing. > > However, it is weird that there was no protein expression either in > the soluble fraction or as inclusion bodies. > > Could anyone give some instruction? > > Thanks a lot and have a nice weekend, > > Jerry > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
