I agree with Engin’s suggestions. Our group crystallized a complex of mesotypsin with BPTI, where we measured an inhibition constant (approximating Kd) of 14 micromolar, by mixing the two pure proteins together in 1:1 stoichiometry. Actually we do that for a lot of our complexes with higher affinity also. It is definitely worth a try.
http://www.jbc.org/content/283/7/4115.full Evette Evette S. Radisky, Ph.D. Associate Professor and Consultant Department of Cancer Biology Mayo Clinic Cancer Center _________________________________________ Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Engin Özkan Sent: Tuesday, January 31, 2017 1:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] For stabilizing protein-protein complex Not observing a complex on a gel filtration run for a week complex (micromolar) is not necessarily unexpected. It all depends on your protein concentrations, the dissociation kinetics (remember, a gel filtration run is not an equilibrium experiment - it dilutes and separates your sample), etc. I would make sure that the SPR results are believable and sensible: if the affinity is really weak, I'd expect very fast kinetics - most weak interactions come with faster association and dissociation. As a result, in this affinity range, you can't fit kinetics data in SPR, and can only do an Langmuir isotherm fit to your equilibrium binding response values. Also, you should check your expected Rmax values (based on how much you captured on the chip), and make sure that your responses don't exceed the theoretical values. Otherwise, you are just observing non-specific binding. Similarly, if your responses are way too low compared to the expected, your protein on the chip might be mostly inactive - again, not good. If everything checks out, just mix your two proteins at the right stoichiometric ratio, concentrate a lot, and go ahead with crystallization (if that's what you want). Engin On 1/31/17 10:05 AM, sanjeev kumar wrote: Dear all, I am trying to stabilize a protein-protein complex. Our SPR study indicates it is having micro molar dissociation constant. I tried to purify both the molecule in complex form with size exclusion chromatography (mixed both the protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt observed formation of complex as both the molecule eluted at their respected elution volume. Please suggest me to get a better way to achieve the complex and if anyone gives idea about what is the good cross-linker I can use. Suggestions are highly appreciated. Thanks best sanjeev kumar, PhD Purdue University West Lafayette Indiana
