Re: [ccp4bb] Kinase crystallization
a comment to add about phosphatase (PP2A, C class serine theronine) buying phosphatase for crystallization trials was kind of not practical( very high prices ) so we tried expressing and purify it from overexpressed in bacterium with yields very low. Also the purified phosphtase co-purify some proteasaes that led to proteolysis of the samples. what is the experts here experienced in such conditions? Judit, Is there a better source? cheaper and reliable? thanks On Mon, Jan 30, 2012 at 8:44 AM, Debreczeni, Judit judit.debrecz...@astrazeneca.com wrote: 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. 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. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CHAVES SANJUAN, ANTONIO Sent: 30 January 2012 10:07 To: CCP4BB@JISCMAIL.AC.UK 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 -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] Kinase crystallization
It is a fairly common issue with kinases. Among other options you may want to try a generic kinase inhibitor (there are several good ones just look at pdb structures for ieas) and if this does not help then you could attempt to clamp the motion down via an inter-lobe engineered disulphide bond... Artem On Jan 30, 2012 5:17 AM, CHAVES SANJUAN, ANTONIO xanto...@iqfr.csic.es wrote: 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
Re: [ccp4bb] Kinase crystallization
Staurosporine come to mind as a general kinase inhibitor. I also second Artem's suggestion that ligands make a big difference, we had several cases of kinases which required ligands for crystallization success. Also make sure you eliminate any floppy ends which may interfere with packing. Good luck Carsten From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CHAVES SANJUAN, ANTONIO Sent: Monday, January 30, 2012 5:07 AM To: CCP4BB@JISCMAIL.AC.UK 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
Re: [ccp4bb] Kinase crystallization
if the ligand binding site is exposed to the solvent a bound ligand may help. if the protein has is flexible domains and ligand fix it in one conformation, a bound ligand will help even more. All the above assuming that the purity and the concentration of the protein are high. George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debreczeni, Judit Sent: Monday, January 30, 2012 3:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Kinase crystallization 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. _ 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. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CHAVES SANJUAN, ANTONIO Sent: 30 January 2012 10:07 To: CCP4BB@JISCMAIL.AC.UK 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
