TY - JOUR
T1 - Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli
AU - Delli-Bovi, Teegan A.
AU - Spalding, Maroya D.
AU - Prigge, Sean T.
N1 - Funding Information:
Plasmid pCY216 was a gift from John Cronan at the University of Illinois at Urbana-Champaign. Plasmid pDB1282 was a gift from Dennis Dean at Virginia Polytechnic Institute and State University (Virginia Tech). The biotin auxotroph E. coli strain Keio JW0758 was provided by the National BioResource Project (NIG, Japan). Plasmid pRK586 was a gift from David Waugh at the National Cancer Institute. The authors thank Caelin Potts, Jolyn Gisselberg and Swetha Velivela for reagents and technical assistance. This work was supported by the National Institutes of Health (R01 AI065853), the Johns Hopkins Malaria Research Institute and the Bloomberg Family Foundation.
PY - 2010/10/11
Y1 - 2010/10/11
N2 - Background: 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.Results: 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.Conclusions: 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.
AB - Background: 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.Results: 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.Conclusions: 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.
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U2 - 10.1186/1472-6750-10-73
DO - 10.1186/1472-6750-10-73
M3 - Article
C2 - 20937134
AN - SCOPUS:77957659562
VL - 10
JO - BMC Biotechnology
JF - BMC Biotechnology
SN - 1472-6750
M1 - 73
ER -