[ccp4bb] purification
Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien
Re: [ccp4bb] Purification
Hello everyone, Just want to say thanks for your great ideas and time to reply my question. Hope I will solve my problem soon Kien
[ccp4bb] off topic
Hello everyone, I am trying to purify a 13 KDa membrane protein using Ni NTA. The protein is solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and binds very well to the column. However, it also turns the column brownish. The protein contains 4 cysteine residues so I suspect that this causes cross-linking with other proteins and thus brownish precipitation on the column. So I included 5 mM beta-ME in my buffer to prevent disulfide bond formation but this doesn't help. I tried 1 mM DTT and this ruined the column. Help!! Is there anyway to prevent this brownish problem? Thanks a lot in advance Kien
Re: [ccp4bb] How to express a >95KD FAD protein
try codon optimisation use Rosetta 2 strains fuse to MBP I expressed a yeast protein of ~100 KDa, expression was low until I fused it to MBp (resulting protein ~ 150 KDa)
