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Biotinylation of proteins, including antibodies, is a very useful and important modification for a variety of biochemical characterizations, including anti-drug antibody (ADA) assays used to detect antibodies raised against therapeutic antibodies. We assessed different degrees of biotin labeling of an anti-cocaine mAb currently under development for treating cocaine use disorder. We noted that higher levels of biotin labeling dramatically decreased mAb solubility, and increased the tendency to bind to surfaces, complicating characterization of the biotinylated antibody. Specifically, in phosphate buffered saline, labeling stoichiometries of more than about 3 biotin/mAb resulted in decreased recoveries due to increased binding to surfaces and decreased mAb solubility. Native gel agarose electrophoresis, differential scanning fluorimetry, and isothermal titration calorimetry all demonstrated changes in the mAb which became more pronounced above a labeling ratio of 3 biotin/mAb. At 3.0 biotin/mAb, there were minimal changes in solubility, adsorptivity, exposure of hydrophobic dye-binding sites, heat stability, and cocaine binding, in stark contrast to labeling with 5.6 biotin/mAb. Thus, the degree of biotinylation should be kept at about 3 biotin/mAb to maintain antigen binding and general structure, solubility, and stability of this mAb, a finding which may be important for other similar mAbs currently in use or under development. [Display omitted] • Anti-cocaine h2E2 mAb was biotinylated at levels from about 2 to 8 biotin/mAb. • Increasing biotinylation greatly reduced mAb solubility in PBS buffer. • At >3 biotin/mAb, reversible mAb adsorption caused variation in measured biotin/mAb. • DSF revealed large increases in hydrophobic mAb binding of Sypro dye at >3 biotin/mAb. • ITC revealed only small changes in mAb cocaine (antigen) binding at >3 biotin/mAb. [ABSTRACT FROM AUTHOR]

Src-homology-2-domain-containing protein tyrosine phosphatase-2 (SHP2) is a signaling enzyme whose activity is governed by an equilibrium between autoinhibited and activated states. Regulation of SHP2 activity is critical for cellular homeostasis, and mutations that alter its autoregulatory equilibrium cause cancers and developmental disorders. Several methods for assessing the strength of autoinhibitory interactions in SHP2 mutants have been previously reported, but each has limitations. We show that differential scanning fluorimetry provides a rapid, quantitative measure of SHP2 autoinhibition that is independent of the intrinsic activity of the SHP2 mutant being analyzed, does not involve protein labeling, and does not require specialized instrumentation. [Display omitted] • SHP2's activity is controlled by an interdomain autoinhibitory interaction. • Mutations that weaken SHP2's autoinhibition are causative for human pathologies. • Assessing SHP2 autoinhibition generally requires specialized equipment. • Differential scanning fluorimetry provides a rapid measure of SHP2 autoinhibition. • DSF does not require protein labeling or specialized instrumentation. [ABSTRACT FROM AUTHOR]

The use of enzymes in organic solvents has considerably widened their repertoire of applications. Such low water containing media also offer the possibility of carrying out enzymatic reactions at higher temperatures and enhancing reaction yields. The utility of such preparations is limited by the damage caused to the protein structure during freeze-drying. This work investigates the result of exposing the proteolytic enzyme subtilisin to high temperature in low water containing n-octane on its activity in aqueous and non-aqueous media. Exposing subtilisin at 90 °C for 5 h led to 18-fold improvement in its transesterification activity even at the normal assay temperature (37 °C) when compared with the untreated enzyme. The use of n-octane as the reaction medium was important as it helped to retain the three-dimensional architecture of the enzyme and should be considered while designing strategies for obtaining high activity preparations of other enzymes. Structural analysis using differential scanning fluorimetry showed that the enzyme lost its structure after heating in aqueous medium but retained it when heated in organic solvent. The simplicity and general applicability of the strategy should make it useful for obtaining highly active preparations of other enzymes as well. [ABSTRACT FROM AUTHOR]

The K i of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO 2 hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO 2 and the inability to determine the K i below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K d below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO 2 concentration were developed for the determination of k cat and K M of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor K i . The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the K d of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly overexpressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design. [ABSTRACT FROM AUTHOR]

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding ( K nd ), and protein unfolding by urea ( I 50 ). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity. [ABSTRACT FROM AUTHOR]

Abstract: A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT–PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins. [Copyright &y& Elsevier]

Abstract: Thermal shift analysis is becoming widely used as a method to identify initial hit ligands for inhibitor discovery or to identify ligands that may aid crystallization. The data analysis software provided by the equipment manufacturers or in the public domain is cumbersome to use. We have assessed a number of different approaches to generate a value for the melting temperature (T m) and implemented these methods in the program MTSA within the commercial software Matlab to provide an easy-to-use and rapid way to process experimental thermal shift data. The program outputs the T m, the quality of the fit, and the deviation from a standard value, the thermal shift ΔT m. Our analysis of these results includes a discussion of some issues with previous publications in this area. We conclude that the most suitable value for T m should be taken from the midpoint determined for a curve fitted to the experimental data with a five-parameter equation. In addition, we found that different ranking of ligand binding can be obtained using the different techniques when screening for binding of weak ligands such as fragments. Therefore, the technique should be used with caution for such screening. [Copyright &y& Elsevier]

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the K d values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT–PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL l -cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT–PCR machines in a hydrophilic or hydrophobic environment. [ABSTRACT FROM AUTHOR]

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔT m , while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization. Image 1 • Three assays for the evaluation of binding affinities of TRF2 inhibitors were explored. • 1:1 kinetic binding sensorgrams were obtained from label-free BioLayer Interferometry (BLI). • K d values of the reference compound from BLI assays consist with K i values from competitive FP assays. [ABSTRACT FROM AUTHOR]

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology. Image 1 • VHH are small antibody-like proteins. They are entering the therapeutic arsenal. • VHH purity, homogeneity, structure and affinity towards their targets are described. • An analytical workflow to qualify those proteins is presented to validate the molecules. [ABSTRACT FROM AUTHOR]