PubMed:20937134
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/20937134","sourcedb":"PubMed","sourceid":"20937134","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/20937134","text":"Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli.\nBACKGROUND: Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.\nRESULTS: In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.\nCONCLUSIONS: The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.","tracks":[{"project":"PubMed_Structured_Abstracts","denotations":[{"id":"T1","span":{"begin":141,"end":815},"obj":"BACKGROUND"},{"id":"T2","span":{"begin":825,"end":1579},"obj":"RESULTS"},{"id":"T3","span":{"begin":1593,"end":1748},"obj":"CONCLUSIONS"}],"attributes":[{"subj":"T1","pred":"source","obj":"PubMed_Structured_Abstracts"},{"subj":"T2","pred":"source","obj":"PubMed_Structured_Abstracts"},{"subj":"T3","pred":"source","obj":"PubMed_Structured_Abstracts"}]},{"project":"Allie","denotations":[{"id":"SS1_20937134_9_0","span":{"begin":1072,"end":1094},"obj":"expanded"},{"id":"SS2_20937134_9_0","span":{"begin":1096,"end":1099},"obj":"abbr"}],"relations":[{"id":"AE1_20937134_9_0","pred":"abbreviatedTo","subj":"SS1_20937134_9_0","obj":"SS2_20937134_9_0"}],"attributes":[{"subj":"SS1_20937134_9_0","pred":"source","obj":"Allie"},{"subj":"SS2_20937134_9_0","pred":"source","obj":"Allie"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"PubMed_Structured_Abstracts","color":"#93c6ec","default":true},{"id":"Allie","color":"#e0ec93"}]}]}}