We recently trying to express scfv-his tagged through Cytoplasmic expressions using SHUFFLE strain. We are getting a high protein expression related to 50 kDa, instead of ~27 (our target). This 50kDa also getting Purified in histrap.
We initially thought that this is the Dimeric form. But even after strong treatment With SDS and beta mercapo, this 50 kDa remains intact.
And very very mild band of our target protein is observed.
Anyone experiences this issue? Any suggestions please ?
am not sure the purpose of ur question.
・For purify the sample, size exclusion chromatography ( HiLoad 16/600 Superdex 75 pg prep grade (GE Healthcare Bioscience)) could help;
・For increase the expression level of ur target scFv, I suggest co-expression with chaperone (doi: 10.1093/jb/mvz059), change the expression vector or fuse the solubilization tag (e.g. MBP tag );
・If you just want to analyze the 50 kDa band, maybe you can try western blot at first.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression