Does your kinase autophosphorylate by any chance? -- That can produce differently phosphorylated species and affect crystallisability. You can detect it by e.g. mass spec, and tackle it by dephosphorylating the protein prior to crystallisation or by coexpression with a phosphatase.
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of CHAVES SANJUAN, ANTONIO Sent: 30 January 2012 10:07 To: [email protected] Subject: [ccp4bb] Kinase crystallization Dear all, I am trying to crystallize a protein kinase without any success. I suspect about its characteristic catalytic loop. I have already prepared different constructs, different expression vectors, and different mutant proteins (pseudo-phosphorylated, active, inactive?). I have also tested some ligands as ANPpnp (non hidrolizable nucleotide), ADP (product) and manganese (cofactor). I am thinking in trying to cocrystallize it with a general kinase substrate (a peptide, a small molecule...). Does any one have any experience or suggestion? Thanks in advance. Sincerely, Antonio -------------------------------------------------------------------------- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies.
