Proteins biotinylation

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

Add a quantity of the biotin-PEG -amine solution to the solution containing the car-boxylate molecule to achieve the desired molar excess. For molecules containing a single carboxylate to be modified, a 1.5- to 2-fold molar excess may be sufficient. However, for proteins or peptides that also contain competing amines, a much larger excess of biotin compound should be used (e.g., 100-fold excess). For instance, for protein biotinylation, add 120 pi of the biotin-PEG -amine solution per ml of the solution prepared in Step 1. 

LaRochelle, W.J., and Froehner, S.C. (1986b) Immunochemical detection of proteins biotinylated on nitrocellulose replicas./. Immunol. Meth. 92, 65-71. 

Protein biotinylation is catalyzed by biotin protein ligase (BPL). In the active site of the enzyme, biotin is activated at the expense of ATP to form AMP-biotin the activated biotin can then react with a nucleophile on the targeted protein. BPL transfers the biotin to a special lysine on biotin carboxyl carrier protein (BCCP), a subunit of AcCoA carboxylase (Scheme 21). Biotinylation of BCCP is very important in fatty acid biosynthesis, starting the growth of the fatty acid with AcCoA carboxylase to generate malonyl-CoA. Recently the crystal structures of mutated BPL and BCCP have been solved together with biotin and ATP to get a better idea of how the transfer fiinctions. ... 

Chattopadhaya S, Tan LP, Yao SQ. Strategies for site-specific protein biotinylation using in vitro, in vivo, and cell-free systems toward functional protein arrays. Nat. Rotoc. 2006 1 2386-2398. 

There are several methods to assess degree of protein biotinylation. For example, titration of protein amino groups with TNBS before and after biotinylation allows estimation of the total number of biotin residues coupled per protein molecule (12). However, only streptavidin-accessible biotin residues localized on the surface of biotinylated protein contribute to its conjugation with streptavidin. To determine such accessible biotin residues we utilize direct solid-phase radioassay of binding of radiolabeled streptavidin to immobilized biotinylated protein (or vice versa) (17). 

Lue RYP, Chen GYJ, Qing Zhu YH, Yao SQ (2003) Versatile protein biotinylation strategies for potential high-throughput proteomics. J Am Chem Soc 126 1055-1062... 

Bayer Diagnostics 2008 Roche 2008). One way to do this is to couple the binding protein covalently to solid-phase particles in the form of paramagnetic beads. Another approach is to use biotinylated cobalamin as the competitive agent after binding to the protein, biotinylated cobalamin is precipitated onto streptavidin linked to solid-phase particles. In either case, the protein-cobalamin equilibrium may be disturbed in washing steps, and leakage from the solid phase may also be an issue to consider especially if these analytical principles are used in in-house assays. 

Tissot, G., Job, D., Douce, R. and Alban, C. (1996) Protein biotinylation in higher plants Characterization of biotin holocarboxylase synthetase activity from pea leaves, Biochem. J. 314, 391-395. 

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