Biotin carboxyl carrier protein co-purifies as a contaminant in core-streptavidin preparations.
We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80 degrees C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.
Wang
Welson Wen-Shang
WW
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 114 St - 89 Ave, Edmonton, Alberta, Canada T6G 2N8.
Das
Dipankar
D
Suresh
Mavanur R
MR
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification*; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II; D066298:In Vitro Techniques; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D019809:Streptavidin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein