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PubMed Journal Database | Protein engineering, design & selection : PEDS RSS

05:24 EDT 16th June 2019 | BioPortfolio

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Showing PubMed Articles 1–25 of 66 from Protein engineering, design & selection : PEDS

High-throughput reformatting of phage-displayed antibody fragments to IgGs by one-step emulsion PCR.

Single-chain variable fragment (scFv) is the most common format for phage display antibody library. The isolated scFvs need to be reformatted to full-length IgGs for further characterization. High throughput reformatting of scFv to IgG without disrupting VH-VL pairing is of great demanding for exhaustive screening of all antibodies in IgG format. Herein, we developed a strategy based on the overlap extension PCR in emulsion to reformat scFv to IgG while maintain the accuracy and complexity of variable regio...

Insight into the aggregation of lipase from Pseudomonas sp. using mutagenesis: protection of aggregation prone region by adoption of α-helix structure.

Previously, a lipase purified from a Pseudomonas source showed to form amyloid fibril structure very rapidly in the absence of a detectable lag phase. In this process the urea-unfolded enzyme encounters a medium close to physiological, but is unable to fold and, therefore, the main driving force of aggregation lies in the sequence of the protein and in its aggregation-promoting regions (APRs). Two regions with the highest propensity to aggregate were identified. These were Regions 51-57 and 160-172 as they ...

Computational-guided determination of the functional role of 447-52D long CDRH3.

447-52D (447) is a human monoclonal antibody that recognizes a conserved epitope in the crown region of the third variable loop (V3) of HIV-1 gp120, and like many anti-HIV-1 antibodies with broad neutralization capabilities, it has a long heavy-chain complementarity determining region (CDRH3). Here, we use a combination of computational mutagenesis and modeling in tandem with fluorescence polarization assays to interrogate the molecular basis of 447 CDRH3 length and the individual contribution of selected C...

Net charge of antibody complementarity-determining regions is a key predictor of specificity.

Specificity is one of the most important and complex properties that is central to both natural antibody function and therapeutic antibody efficacy. However, it has proven extremely challenging to define robust guidelines for predicting antibody specificity. Here we evaluated the physicochemical determinants of antibody specificity for multiple panels of antibodies, including >100 clinical-stage antibodies. Surprisingly, we find that the theoretical net charge of the complementarity-determining regions (CDR...

Tag-on-Demand: exploiting amber codon suppression technology for the enrichment of high-expressing membrane protein cell lines.

Membrane proteins play key roles in the evolution of numerous diseases and as a result have become the most dominant class of targets for therapeutic intervention. However, their poor expression and detection oftentimes prohibit drug discovery and screening efforts. Herein, we have developed an approach, named 'Tag-on-Demand' that exploits amber suppression to control the expression of 'tagged' membrane proteins for detection and selections, yet can be turned off for expression of the protein in its native ...

Seeking allosteric networks in PDZ domains.

Ever since Ranganathan and coworkers subjected the covariation of amino acid residues in the postsynaptic density-95/Discs large/Zonula occludens 1 (PDZ) domain family to a statistical correlation analysis, PDZ domains have represented a paradigmatic family to explore single domain protein allostery. Nevertheless, several theoretical and experimental studies in the past two decades have contributed contradicting results with regard to structural localization of the allosteric networks, or even questioned th...

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development o...

Unintended specificity of an engineered ligand-binding protein facilitated by unpredicted plasticity of the protein fold.

Attempts to create novel ligand-binding proteins often focus on formation of a binding pocket with shape complementarity against the desired ligand (particularly for compounds that lack distinct polar moieties). Although designed proteins often exhibit binding of the desired ligand, in some cases they display unintended recognition behavior. One such designed protein, that was originally intended to bind tetrahydrocannabinol (THC), was found instead to display binding of 25-hydroxy-cholecalciferol (25-D3) a...

The generation and biological activity of a long-lasting recombinant human interferon-λ1.

The previously generated recombinant human (rh) interferon (IFN)-λ1 protein has a short half-life, and this feature makes it challenging to conduct studies on potential clinical applications for rhIFN-λ1. In an attempt to overcome this difficulty, we constructed a 'long-life' version of rhIFN-λ1. This modified rhIFN-λ1, named rhIFN-λ1-CTPON, has a human chorionic gonadotropin β subunit carboxyl-terminal peptide (CTP) and an N-glycosylation sequence linked to its C-terminus. We confirmed the sequence o...

CDB-a database for protein heterodimeric complexes.

Crystallographic structures of protein complexes are essential to develop proteomic and structural biology methods, as prediction of protein-protein interaction (PPI) sites and protein-protein docking. Such structures can aid the development of protein complexation inhibitors. Complex DataBase (CDB), accessible at www.jct-bioinfo.com/cdb/search, is a database web application for heterodimeric protein crystallographic complexes along with the crystallographic structures of each individual unbound protein. Di...

Antibody discovery and the arrow of time.

Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.

Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduc...

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9...

Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtra...

Rational optimization of a monoclonal antibody for simultaneous improvements in its solution properties and biological activity.

Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutation...

Variable heavy-variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly.

Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers...

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for o...

Analysis of nanobody paratopes reveals greater diversity than classical antibodies.

Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb-antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural f...

Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen.

Structure-based approaches to antigen design utilize insights from antibody (Ab):antigen interactions and a refined understanding of protective Ab responses to engineer novel antigens presenting epitopes with conformations relevant to eliciting or discovering protective humoral responses. For human immunodeficiency virus-1 (HIV-1), one model of protection is provided by broadly neutralizing Abs (bnAbs) against epitopes present in the closed prefusion trimeric conformation of HIV-1 envelope glycoprotein, suc...

The structure of SALM5 suggests a dimeric assembly for the presynaptic RPTP ligand recognition.

Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently, several families of leucine-rich repeat (LRR) domain-containing neuronal adhesion molecules have been characterised, including netrin-G ligands, LRRTMs and the synaptic adhesion-like molecule (SALM) family proteins. Most of these are expressed at the excitatory glutamatergic synapses, and dysfunctions of these genes are genetically linked with cognitive disorders, such as autism spectrum disor...

Creation of a formate: malate oxidoreductase by fusion of dehydrogenase enzymes with PEGylated cofactor swing arms.

Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited c...

Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in One-Step RT-PCR assay.

Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display t...

Identification of high affinity HER2 binding antibodies using CHO Fab surface display.

Discovery of monoclonal antibodies is most commonly performed using phage or yeast display but mammalian cells are used for production because of the complex antibody structure, including the multiple disulfide bonds and glycosylation, required for function. As this transition between host organisms is often accompanied by impaired binding, folding or expression, development pipelines include laborious plate-based screening or engineering strategies to adapt an antibody to mammalian expression. To circumven...

Affinity maturation of an TpoR targeting antibody in full-length IgG form for enhanced agonist activity.

It has been observed that converting scFv formatted antibodies to full-length IgG often associates with loss of affinity. We aim to address this issue in this paper by establishing an integrated affinity maturation method applying yeast display technology platform. To demonstrate that, we employed a human thrombopoietin receptor targeting antibody named 3D9 which was identified previously from a combinational antibody library in scFv-Fc fusion protein form. We have observed that significant potency loss hap...

Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an...


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