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The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.

In order to study the effects carbohydrates have on glycoprotein structure and function, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in ViVo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in Vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a "tag" to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosaccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN′. These fragments were religated by subtilisin 8397 to the full length RNase by addition of glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations.

We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(L-lysine)-grafted poly-(ethylene glycol) copolymers adsorbed on negatively charged surfaces. As a model system, we synthesized biotin-derivatized poly(L-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-[(PEGm) (1؊x) (PEG-biotin)x], where x varies from 0 to 1. Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy. The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures. The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins. This streptavidin layer can subsequently capture biotinylated proteins. Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning. C ontrolling immobilization of biomolecules on surfaces, while preventing nonspecific adsorption of unwanted species, has become an important goal for monitoring specific biointeractions and binding of biomolecules or cells. Indeed, in diagnostic assays, biomaterial devices, and surface-related bioanalytical applications, nonspecific protein binding can often be the obstacle to higher sensitivity, reproducibility, or implant integration. Therefore, in past decades, many immobilization strategies have been established. These include physisorption to solid organic or inorganic supports (noncovalent coupling occurs by electrostatic and van der Waals forces), noncovalent chemisorption, and covalent immobilization on organic thin films of different molecular organization.Physisorption of biomolecules directly on the surface of inorganic substrate materials such as glass or organic coatings such as polymeric materials and adhesion layers (polylysine and nitrocellulose) probably constitutes the least technically challenging immobilization procedure. For instance, spotted microarrays of nucleic acids and, more recently, proteins are mostly based on physical adsorption (1). However, these methods suffer from some key limitations, such as their lack of control over the quantity and orientation of adsorbed biomolecules, and, hence, from lower reproducibility, lower interaction efficiencies, and high error rates. Moreover, additional passivation or blocking steps of the remaining sites are often required to limit the extent of nonspecific binding and protein denaturation. These are serious limitations for protein microarray applications.Attempts to control the biomolecular density and orientation of biomolecules at the solid-liquid interface to obtain better reproducibility have been undertaken through various strategies of covalent and site-specific immobilization. These include mostly immobilization via organic thin films such as selfassembled alkanethiol a...

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To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

This paper describes the solution- and solid-phase synthesis of glycopeptides containing a GlcNAc moiety at different positions and investigates them as substrates for subtilisin-catalyzed glycopeptide condensation, with an aim to develop enzymatic synthesis of complex glycopeptides and glycoproteins. A systematic study was performed to determine the sites at which the protease subtilisin will accept a glycosylated residue in enzymatic peptide ligations. It was found that Fmoc-glycopeptide esters with unprotected side chains can be prepared by solid-phase methodology using the Rink amide resin. Both removal of the side chain protecting groups as well as release from the resin was accomplished by treatment with acid to provide glycopeptide esters of the benzyl type which were used directly as substrates. One such 12 residue peptide ester was used in a subtilisin-catalyzed glycopeptide condensation in a kinetically controlled mode to demonstrate the utility of this approach.

Several enzymes immobilized on thermo‐responsive polyacrylamide polymers are nearly as active as their soluble forms, and can be recovered for reuse after gentle heating and precipitation. Carbohydrates attached to these polymers have been used for enzymatic glycosylation, and the products have been isolated by thermal precipitation followed by release from the polymer, thus greatly simplifying product purification in water.