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
