[ccp4bb] pH dependent conformational change

Dear CCP4 colleagues,   We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure c

[ccp4bb] off topic, design of a self-cleaving tag

Hi, We would like to design a self-cleaving tag. It will be similar to the one Roger Tsien has developed but I hope to find one that works in the opposite way. Lin, M.Z. et al. A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals. Proc. Natl. Acad. Sci

[ccp4bb] pH sensitive fluorescent dye

Hi, Sorry for the off-CCP4 question. Crystallographers are now doing everything... We are looking for a fluorescent dye that will be turned on, or increase signal, when pH goes from neutral to acidic. More specifically, we would like to monitor the internalization of a protein into the ac

[ccp4bb] low insect cell expression

Hi,   We are trying to express a protein in insect cells as secreted. However, the yield is VERY low using either pFastBAC or pAcGP67A. We  have tried many different constructs and this is the only one that at least expresses. I wonder whether anyone knows how to improve. Many thanks.   Best, Ch

[ccp4bb] Centramate

Hi,   Sorry for the off topic question. I am looking for helps from someone who is familiar with Centramate from Pall. We recently bought one and try to use it for concentrating insect media with secreted proteins. Here is some more information:   Pump: MasterFlex Console drive Pump head: quic

[ccp4bb] peptide for co-crystallization

Hi,   We would like to order two peptides for co-crystallizaiton. I notice that there is a big price jump for peptide over 95% pure and over 98% pure. Do you think 95% purity is good enough? Another choice is to order crude peptides and purify by ourselves. Crude peptide is much cheaper. But we

[ccp4bb] Off topic: insect cell expression

Hi there,   Sorry for the off topic questions. We need your feedback.   We are expressing a rat protein in insect cells. It is expressed as a secreted protein with an N-terminal 6xHis tag. We can get about 4 mg of it from 1L culture and everything looked quite normal at the very beginning (at 4C)

[ccp4bb] protein expression

Hi, I have a protein with two independently folded domains. I can express either one in bacteria with pretty good expression yield. However, when I put them together with a linker, the expression drops significantly. I can barely see any soluble protein and most of it is now inclusion bodi

[ccp4bb] protein expression problem

Hi, I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM

[ccp4bb] how to change a membrane protein into a water soluble protein?

Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank

[ccp4bb] water soluble protein that needs detergent to be stable

Hi, I am working on a 60 kDa C. elegans protein that is predicted to be mostly alpha-helix. It is over-expressed in E.coli and the yield is about 1 mg/L of cell culture. The CD spec at 4 degree showed the presence of dominant alpha-helix. However, we don’t have any functional assay to conf