NHS-PEG„-biotin

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

Dissolve the NHS-PEG -biotin compound in DMAC, DMSO, or DMF (pure and dry) at a concentration of 10-20 mM. Prepare fresh in a fume hood. 

With mixing, add an aliquot of the biotinylation stock solution to the protein solution to provide at least a 10-fold molar excess over the concentration of protein present. Doing a series of reactions with different molar amounts of the NHS-PEG -biotin compound may be done to optimize the modification level. 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

Danion et al. immobilized intact liposomes onto SCLs (Figure 51.11). In the first step, poly-ethylenimine was covalently bounded onto the hydroxyl groups available on the surface of a commercial CL (Hioxifilcon B). Then, NHS-PEG-biotin molecules were bounded onto the surface amine groups by carbodiimide chemistry. NeutrAvidin were bounded onto the PEG-biotin layer. Liposomes containing PEG-biotinylated lipids were docked onto the surface-immobilized NeutrAvidin. Consecutive addition of further NeutrAvidin and liposome layers enabled the fabrication of multilayers. Multilayers of liposomes were also produced by exposing CLs coated with NeutrAvidin to liposome aggregates produced by the addition of free biotin in solution. 

The biotinylation of amine-dendrimers may be accomplished using either an organic reaction environment or an aqueous medium. For modification of PAMAM dendrimers with a biotinidase resistant biotin compound, Wilbur et al. (1998) performed the reaction in DMF with triethylamine as catalyst (proton acceptor). The following protocol illustrates this type of procedure using the biotinylation reagent NHS-PEG/pbiotin, which closely compares to the biotinidase insensitive compound used in the published procedure. 

Although NHS-LC-biotin and sulfo-NHS-LC-biotin are very popular reagents for biotinylation, they both result in hydrophobic aliphatic biotin modifications on proteins and antibodies. Unfortunately, these groups have a tendency to aggregate in aqueous solution and may cause protein precipitation or loss of activity over time. For this reason, the use of more hydrophilic PEG-based biotin compounds of approximately the same spacer length may be a better alternative for maintaining water solubility of modified proteins (Chapter 18). 

Hydrophilic short biotin-PEG tags also have found their way into the design of multifunctional crosslinkers to study protein structures by mass spec. Fujii et al. (2004) developed a homobifunctional NHS ester crosslinker that in addition has a PEG-biotin handle (Figure 18.1). The reagent actually is a trifunctional compound similar to the biotinylated PIR compound... 

However, since many of the traditional biotinylation reagents, such as NHS-LC-biotin contain hydrophobic spacers, their use with amphipathic liposomal constructions may not be entirely appropriate. A better choice may be to use a hydrophilic PEG-based biotin compound that creates a water-soluble biotin modification on the outer aqueous surface of the liposome bilayer. Biotinylation reagents of this type are discussed in Chapter 18, Section 3. 

Figure 28.6 A trifunctional PIR compound that contains two NHS esters to capture interacting proteins through amide bond formation and a PEG-biotin arm to permit isolation of crosslinked proteins on (strept)avidin supports.

If desired nonnatural amino acids can be incorporated into the peptide. We routinely incorporate Fmoc-NH-(PEG)n-COOH (C42H65N016) (Polypure, Oslo, Norway), Fmoc-Glu(biotinyl-PEG)-OH (C40H55N5O10S), and Fmoc-Lys(biotin)-OH (C31H38N406S) (Novabiochem, EMD Biosciences, San Diego, CA) (see Notes 5 and 6). 

Fmoc-NH-(PEG)n-COOH, Fmoc-Glu(biotinyl-PEG)-OH and Fmoc-Lys(biotin)-OH are coupled in the same fashion as the natural Fmoc-protected amino acids. 

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

Figure 18.15 NHS-chromogenic-PEG3-biotin contains an amine-reactive NHS ester that can be used to label biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of the reagent allows the degree of biotinylation to be quantified by measuring its absorbance at 354 nm. The compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

Figure 18.22 Biotin-PEG -amine can be used to add a biotin label to carboxylate-containing molecules using the EDC/(sulfo)NHS reaction, which forms a stable amide linkage.

Synthesis of Biotin-Derivatized PLL- -PEG. The biotin-derivatized PLL- -PEG was synthesized in a manner similar to that described in the previous section. A stoichiometric amount of biotin-PEG-C02-NHS powder was slowly added to the filtered PLL-HBr solution and stirred vigorously for 1 h. Addition of the corresponding stoichiometric amount of MeO— PEG- SPA powder then followed, and the resulting solution was... 

---