Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

Chattopadhyay, Gopinath; Varadarajan, Raghavan

doi:10.1002/pro.3622

Cited by 45 publications

(65 citation statements)

References 30 publications

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“…The temperature response of AMBN proteins was studied by CD spectrometry and DSF. DSF is commonly used to determine the melting temperature of denaturated protein [47]. In our case, we addressed a question of disintegration of the oligomeric state of AMBN.…”

Section: Discussionmentioning

confidence: 99%

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

Vetyskova

Zouharová

Bednářová

et al. 2020

IJMS

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Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel—the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN—its oligomerization ability—is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

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Section: Discussionmentioning

confidence: 99%

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

Vetyskova

Zouharová

Bednářová

et al. 2020

IJMS

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“…Thermal unfolding experiments were performed using nanoDSF of different proteins as previously described (Teale and Weber, 1957;Bruce et al, 2019;Chattopadhyay and Varadarajan, 2019;Magnusson et al, 2019). nanoDSF, measures the changes in the fluorescence of the intrinsic fluorophores tryptophan and tyrosine upon protein unfolding in a label free manner.…”

Section: Nanodsf Sec-mals and Spr Studiesmentioning

confidence: 99%

VapC21 Toxin Contributes to Drug-Tolerance and Interacts With Non-cognate VapB32 Antitoxin in Mycobacterium tuberculosis

et al. 2020

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The prokaryotic ubiquitous Toxin-antitoxin (TA) modules encodes for a stable toxin and an unstable antitoxin. VapBC subfamily is the most abundant Type II TA system in M. tuberculosis genome. However, the exact physiological role for most of these Type II TA systems are still unknown. Here, we have comprehensively characterized the VapBC21 TA locus from M. tuberculosis. The overexpression of VapC21 inhibited mycobacterial growth in a bacteriostatic manner and as expected, growth inhibition was abrogated upon co-expression of the cognate antitoxin, VapB21. We observed that the deletion of vapC21 had no noticeable influence on the in vitro and in vivo growth of M. tuberculosis. Using co-expression and biophysical studies, we observed that in addition to VapB21, VapC21 is also able to interact with non-cognate antitoxin, VapB32. The strength of interaction varied between the cognate and non-cognate TA pairs. The overexpression of VapC21 resulted in differential expression of approximately 435 transcripts in M. tuberculosis. The transcriptional profiles obtained upon ectopic expression of VapC21 was similar to those reported in M. tuberculosis upon exposure to stress conditions such as nutrient starvation and enduring hypoxic response. Further, VapC21 overexpression also led to increased expression of WhiB7 regulon and bacterial tolerance to aminoglycosides and ethambutol. Taken together, these results indicate that a complex network of interactions exists between non-cognate TA pairs and VapC21 contributes to drug tolerance in vitro.

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“…There are increasing evidences from research groups to show that differential scanning fluorimetry (DSF) is routinely used for determining conformational and colloidal stability of antibody-based therapeutics. In particular, measuring ΔG u of unfolding is an invaluable tool for selecting appropriate mAb formulations and predicting long term storage stability 12,21,22 . The data reported from DSF is the average of n = 3 replicates that verified the precision and accuracy of the technique (ΔG u is +2 kJ/mol and c50 is +0.2 M).…”

Section: Discussionmentioning

confidence: 99%

In Pursuit of Stability Enhancement of a Prostate Cancer Targeting Antibody Derived from a Transgenic Animal Platform

Venkataramani

Ernst

Derebe

et al. 2020

Sci Rep

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Accelerated timelines necessitate the discovery of fully human antibodies as biotherapeutics using transgenic animals with a notion that such mAbs bypass humanization. A transgenic animal derived mAb (PCa75) targeted against a prostate cancer antigen had several 'unusual residues' (rare somatic hypermutations, rSHM, with positional frequency of <1%) that resulted in compromised biophysical properties (tm = 61 °C and intrinsic stability ΔGu = 24.3 kJ/mol) and a sub-optimal immunogenicity profile. In our quest for quality medicine, we pursued antibody engineering strategies to enhance the stability of PCa75. PCa62, an engineered variant of PCa75, retained function while significantly improving the drug-like attributes of the molecule (Tm = 75 °C and intrinsic stability ΔGu = 63.5 kJ/ mol). rSHM is rather prevalent, 18 out the 21 approved transgenic animal-derived antibodies have at least one 'unusual residue'. Thus, engineering of rSHM remains critical to enhance the stability and minimize immunogenicity risk of biotherapeutics. Monoclonal antibodies (mAbs) have fundamentally transformed the treatment of complex diseases like autoimmune disorders, cancer, and others over the last two decades 1. The complex physicochemical makeup of these biologics compared to traditional small molecule drugs is manifested in development challenges associated with immunogenicity, aggregation, chemical stability, and physical stability during drug production and delivery. These challenges are outweighed by the unmatched specificity, potency, and safety of these molecules such that mAbs remain a growing source of therapeutic molecules, particularly in Oncology applications, with over 100 new mAbs entering clinical trials yearly 2. With over 570 mAbs currently being tested in clinical trials with overlapping targets, mechanisms, and disease indications, optimization of a drug's physical attributes can result in a significant competitive advantage while also providing better value to patients. Humanized antibodies represent ~43% (i.e. 38 mAbs) of the 89 FDA approved antibodies 2. Although once tedious and time consuming, synthetic biology breakthroughs, sequencing technologies, and worldwide services for high-throughput antibody engineering have drastically reduced the time required for humanization. More recently several transgenic animal platforms have been developed to discover fully human therapeutic antibodies in rodents, circumventing the iterative process of humanization 3,4. Indeed, 21 biotherapeutics derived from four different transgenic animal platforms such as UltimAb, Xenomouse, VelocImmune and TransChromo platforms have been approved and marketed while antibodies from other transgenic animals' platforms like Merus, Harbour Biomed, Ablexis, Wuxi Pharma, Open Monoclonal Technology among others are currently in clinical trials 5. Recreating the human immune system in transgenic animals is challenging as antibody genes are assembled by V-D-J recombination and further diversified by somatic hyper-mutations to enhance specificit...

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Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

Cited by 45 publications

References 30 publications

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

VapC21 Toxin Contributes to Drug-Tolerance and Interacts With Non-cognate VapB32 Antitoxin in Mycobacterium tuberculosis

In Pursuit of Stability Enhancement of a Prostate Cancer Targeting Antibody Derived from a Transgenic Animal Platform

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