Dear Sanjeev, You can try to clone both of your gene in pET duet vector to coexpress both proteins in bacterial expression system and purify with Nickel affinity exchange chromatography (both proteins will be his tagged). This will eliminate the problem of critical mixing of both proteins in stochiometric ratio as in vivo condition (i.e. bacterial cell) will take care of that. Other wise, you may determine the stochiometric binding ratio from SPR data (like by using Langmuir or other models) and mix the individual proteins accordingly. Buffer (and ions) play some crucial role in some protein protein interaction. So you may also try to play around with different buffer and salt compositions (if you intuitively guess the ion or buffer conditions). As the Kd value is in micro molar range, it seems it is not of very strong affinity kind of interaction. So longer incubation (4 degree/ rotation) might give at least formation of some complex. Hope this helps.
Best!! Debasish Kumar Ghosh CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html ----- Original Message ----- From: sanjeev kumar <sanjeev....@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 31 Jan 2017 21:35:32 +0530 (IST) Subject: [ccp4bb] For stabilizing protein-protein complex 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
