Re: [ccp4bb] E. coli BirA biotin ligase expression/purification
Dear Gloria, I think that the most economical biotinylation manner is in vivo, by co-expression of a tagged protein with BirA. One can PCR amplify the BirA cDNA from E. coli and clone it into plasmids for any expression system and sub-cellular localization needed. Ready to use BirA expression plasmids are also available on Addgene. Some extra biotin has to be added to culture medium in my experience. Examples from our lab (HEK cells) are in PMID: 27418511 and 28817804. Best wishes, Radu > Dear friends in crystallography, > I know this seems unrelated, but it really isn't ... please forgive me. We are trying to use the Avitag/BirA system to specifically biotinylate target proteins in an economical manner. Are any of you purifying the BirA enzyme in your lab for biotinylation and if so can you help me figure out the right plasmid/purification protocol? Please let me know. Thank you. Gloria > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. tel: +44-(0)1223-267049 fax: +44-(0)1223-268305 www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] E. coli BirA biotin ligase expression/purification
Dear Gloria, This is the basic protocol we follow. Hope it helps Arnaud Gautier and Marlon J. Hinner (eds.), Site-Specifi c Protein Labeling: Methods and Protocols, Methods in Molecular Biology, vol. 1266, DOI 10.1007/978-1-4939-2272-7_12, © Springer Science+Business Media New York 2015 Linda From: CCP4 bulletin board on behalf of Gloria Borgstahl Sent: Monday, October 21, 2019 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] E. coli BirA biotin ligase expression/purification ATTENTION: This email originated from a sender outside of MCW. Use caution when clicking on links or opening attachments. Dear friends in crystallography, I know this seems unrelated, but it really isn't ... please forgive me. We are trying to use the Avitag/BirA system to specifically biotinylate target proteins in an economical manner. Are any of you purifying the BirA enzyme in your lab for biotinylation and if so can you help me figure out the right plasmid/purification protocol? Please let me know. Thank you. Gloria To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=aFamLAsxMIDYjNglYHTMV0iqFn3z4pVFYPQkjgspw4Y=iHKIgRWrjiS1dpoNu_8JEA=G-X7Orq-6E-4lIXTlihyWYVCq3HqYZlxVwVvvz-ZdO0=0NRMvIxzXZM_8e_Y3kg7SIMwmj9Y2sR4ol8QPSWeXto=> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] E. coli BirA biotin ligase expression/purification
Dear friends in crystallography, I know this seems unrelated, but it really isn't ... please forgive me. We are trying to use the Avitag/BirA system to specifically biotinylate target proteins in an economical manner. Are any of you purifying the BirA enzyme in your lab for biotinylation and if so can you help me figure out the right plasmid/purification protocol? Please let me know. Thank you. Gloria To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
