Re: [ccp4bb] Expression of large proteins in E. coli

Hi Nick, A couple of other suggestions: (1) Try an E. coli K strain (e.g. JM109). They're different in glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805–820) and in my experience tend to grow more slowly - which seems to help improve yields of large constructs. (2) Opt

Re: [ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex

Hello Anindito, While Nick and Paul have thrown you some great ideas around the TAP-tag theme (although GST may not be ideal as it dimerises itself), I feel that I've read your initial e-mail differently from them. Maybe you could clarify? It sounds like you have a specific tag of ~5kDa in mind

Re: [ccp4bb] KD of dimerization, off topic

Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fi

[ccp4bb] off topic - UV absorbance of phosphotyrosine

Hello folks, I would like to measure the concentrations of proteins containing phosphotyrosines using absorbance at 280nm. I'm wondering about calculating extinction coefficients by the Edelhoch method - as used in ProtParam for example: http://web.expasy.org/protparam/protparam-doc.html Given