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https://www.readbyqxmd.com/read/29330770/decoding-selection-bias-imparted-by-unpaired-cysteines-a-tug-of-war-between-expression-and-affinity
#1
B Vijayalakshmi Ayyar, Stephen Hearty, Richard O'Kennedy
In a recombinant antibody scFv format, the presence of an unpaired cysteine (Cys) is implicated in reduced soluble expression and inefficient presentation in phage display. Compared to other species, antibodies derived from rabbits are more likely to contain this unpaired Cys residue at position 80 (Cys80), when generated in a scFv format. In a screening campaign to isolate rabbit scFv against cardiac troponin I (cTnI), it was found that, a large proportion of isolated cTnI-specific clones contained unpaired Cys80...
January 12, 2018: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/29330364/polypharmacy-through-phage-display-selection-of-glucagon-and-glp-1-receptor-co-agonists-from-a-phage-displayed-peptide-library
#2
Anna Demartis, Armin Lahm, Licia Tomei, Elisa Beghetto, Valentina Di Biasio, Federica Orvieto, Francesco Frattolillo, Paul E Carrington, Sheena Mumick, Brian Hawes, Elisabetta Bianchi, Anandan Palani, Antonello Pessi
A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells...
January 12, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29330023/unraveling-the-differential-structural-stability-and-dynamics-features-of-t7-endolysin-partially-folded-conformations
#3
Meenakshi Sharma, Dinesh Kumar, Krishna Mohan Poluri
BACKGROUND: Characterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5-3, T7L PF states display differential ANS binding characteristics. METHODS: CD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations...
January 9, 2018: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29329326/utilization-of-peptide-phage-display-to-investigate-hotspots-on-il-17a-and-what-it-means-for-drug-discovery
#4
Joey P Ting, Frances Tung, Stephen Antonysamy, Stephen Wasserman, Spencer B Jones, Feiyu F Zhang, Alfonso Espada, Howard Broughton, Michael J Chalmers, Michael E Woodman, Holly A Bina, Jeffrey A Dodge, Jordi Benach, Aiping Zhang, Christopher Groshong, Danalyn Manglicmot, Marijane Russell, Sepideh Afshar
To date, IL-17A antibodies remain the only therapeutic approach to correct the abnormal activation of the IL-17A/IL-17R signaling complex. Why is it that despite the remarkable success of IL-17 antibodies, there is no small molecule antagonist of IL-17A in the clinic? Here we offer a unique approach to address this question. In order to understand the interaction of IL-17A with its receptor, we combined peptide discovery using phage display with HDX, crystallography, and functional assays to map and characterize hot regions that contribute to most of the energetics of the IL-17A/IL-17R interaction...
2018: PloS One
https://www.readbyqxmd.com/read/29326975/identifying-the-substrate-proteins-of-u-box-e3s-e4b-and-chip-by-orthogonal-ubiquitin-transfer
#5
Karan Bhuripanyo, Yiyang Wang, Xianpeng Liu, Li Zhou, Ruochuan Liu, Duc Duong, Bo Zhao, Yingtao Bi, Han Zhou, Geng Chen, Nicholas T Seyfried, Walter J Chazin, Hiroaki Kiyokawa, Jun Yin
E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins...
January 2018: Science Advances
https://www.readbyqxmd.com/read/29321208/engineered-synthetic-antibodies-as-probes-to-quantify-the-energetic-contributions-of-ligand-binding-to%C3%A2-conformational-changes-in-proteins
#6
Somnath Mukherjee, Dionne H Griffin, James R Horn, Shahir S Rizk, Malgorzata Nocula-Lugowska, Magnus Malmqvist, Sangwoo S Kim, Anthony A Kossiakoff
Conformational changes in proteins due to ligand binding are ubiquitous in biological systems and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand-binding and those due to structural rearrangements. Here, we used a  unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand-binding to conformation changes...
January 10, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29318563/generation-of-highly-selective-mmp-antibody-inhibitors
#7
Dong Hyun Nam, Xin Ge
Inhibiting individual MMPs of biomedical importance with high selectivity is critical for both fundamental research and therapy development. Here we describe the methods for discovery of inhibitory monoclonal antibodies from synthetic human antibody phage display libraries carrying convex paratopes encoded by long complementarity-determining region (CDR)-H3 segments. We demonstrate the application of this technique for isolation of highly specific and potent antibody inhibitors of human MMP-14.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29315611/isolation-of-a-high-affinity-bet-v-1-specific-igg-derived-scfv-from-a-subject-vaccinated-with-hypoallergenic-bet-v-1-fragments
#8
Elisabeth Gadermaier, Katharina Marth, Christian Lupinek, Raffaela Campana, Gerhard Hofer, Katharina Blatt, Dubravka Smiljkovic, Uwe Roder, Margarete Focke-Tejkl, Susanne Vrtala, Walter Keller, Peter Valent, Rudolf Valenta, Sabine Flicker
BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a non-allergic subject...
January 9, 2018: Allergy
https://www.readbyqxmd.com/read/29311588/characterisation-of-the-antibacterial-properties-of-the-recombinant-phage-endolysins-ap50-31-and-lysb4-as-potent-bactericidal-agents-against-bacillus-anthracis
#9
Sangjin Park, Soo Youn Jun, Chang-Hwan Kim, Gi Mo Jung, Jee Soo Son, Seong Tae Jeong, Seong Jun Yoon, Sang Yup Lee, Sang Hyeon Kang
The recombinant phage endolysins AP50-31 and LysB4 were developed using genetic information from bacteriophages AP50 and B4 and were produced by microbial cultivation followed by chromatographic purification. Subsequently, appropriate formulations were developed that provided an acceptable stability of the recombinant endolysins. The bacteriolytic properties of the formulated endolysins AP50-31 and LysB4 against several bacterial strains belonging to the Bacillus genus including Bacillus anthracis (anthrax) strains were examined...
January 8, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29310289/an-ultrasensitive-electrochemical-immunosensor-for-cry1ab-based-on-phage-displayed-peptides
#10
Xin Lu, Dong-Jian Jiang, Jia-Xiang Yan, Zhen-E Ma, Xiu-Er Luo, Tai-Long Wei, Yang Xu, Qing-Hua He
The rapid and widespread adoption of Bacillus thuringiensis (Bt) proteins in genetically modified (GM) crops has raised concerns about the impact of GM crops on environment and food safety. A sensitive and specific method for detecting Bt proteins in GM crops is of great significance for environment and food risk assessment. In this study, using Cry1Ab as a model Bt protein, an ultrasensitive electrochemical immunosensor for Cry1Ab protein has been developed based on phage displayed peptide. Phage displayed peptides against Cry1Ab protein were obtained from a phage displayed peptide library without animal immunization process through biopanning-elution strategy...
March 1, 2018: Talanta
https://www.readbyqxmd.com/read/29309709/affinity-maturation-of-a-portable-fab-rna-module-for-chaperone-assisted-rna-crystallography
#11
Deepak Koirala, Sandip A Shelke, Marcel Dupont, Stormy Ruiz, Saurja DasGupta, Lucas J Bailey, Steven A Benner, Joseph A Piccirilli
Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity...
January 4, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29306173/blocking-osteopontin-fibronectin-interactions-reduce-extracellular-fibronectin-deployment-and-arthritic-immunopathology
#12
Brij Bhushan Mehta, Saniya Sharma, Rakesh K Vasishta, Ramesh K Sen, Aman Sharma, Manni Luthra-Guptasarma
Elevated levels of a thrombin-cleaved fragment of osteopontin (OPNT) are seen in synovial fluid (SF) and tissues of rheumatoid arthritis (RA) patients. OPNT binds to integrins on cell surfaces, inducing adhesion, migration and survival of inflammatory cells in the synovial joints, where OPNT binds to fibronectin to link fibroblast-like synoviocytes (FLS) with B cells, stimulating the latter to produce inflammatory cytokines. Our aim was to block OPNT-fibronectin interactions and examine whether this reduces inflammation...
January 3, 2018: International Immunopharmacology
https://www.readbyqxmd.com/read/29305748/production-of-human-single-chain-fragment-antibody-scfv-against-human-epidermal-growth-factor-receptor-2-her-2-by-phage-display-technology
#13
Saeideh Foroumadi, Masoumeh Rajabibazl, Azam Rahimpour, Solmaz Shahidi, Walead Ebrahimizadeh, Maral Yarahmadi, Shirin Rajabi, Azam Daraei
Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20-25% on breast cancer tumor cells...
January 5, 2018: In Vitro Cellular & Developmental Biology. Animal
https://www.readbyqxmd.com/read/29304128/identification-of-a-protective-b-cell-epitope-of-the-staphylococcus-aureus-gapc-protein-by-screening-a-phage-displayed-random-peptide-library
#14
Mengyao Wang, Lu Zhai, Wei Yu, Yuhua Wei, Lizi Wang, Shuo Liu, Wanyu Li, Xiaoting Li, Simiao Yu, Xiaoting Chen, Hua Zhang, Jing Chen, Zhenyue Feng, Liquan Yu, Yudong Cui
The impact of epidemic Staphylococcus aureus (S. aureus) on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of S. aureus is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of S. aureus, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response in vivo...
2018: PloS One
https://www.readbyqxmd.com/read/29301517/biopanning-of-polypeptides-binding-to-bovine-ephemeral-fever-virus-g1-protein-from-phage-display-peptide-library
#15
Peili Hou, Guimin Zhao, Chengqiang He, Hongmei Wang, Hongbin He
BACKGROUND: The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication. METHODS: The purified BEFV G1 was coated and then reacted with the M13-based Ph...
January 4, 2018: BMC Veterinary Research
https://www.readbyqxmd.com/read/29301020/discovery-of-internalizing-antibodies-to-basal-breast-cancer-cells
#16
Yu Zhou, Hao Zou, Christina Yau, Lequn Zhao, Steven C Hall, Daryl C Drummond, Shauna Farr-Jones, John W Park, Christopher C Benz, James D Marks
We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies...
December 28, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/29289749/sequence-defined-antibodies-improve-the-detection-of-cadherin-2-n-cadherin-during-zebrafish-development
#17
Giulio Russo, Ulrike Theisen, Wieland Fahr, Saskia Helmsing, Michael Hust, Reinhard W Köster, Stefan Dübel
Cadherin-2 plays a fundamental role during zebrafish CNS and heart morphogenesis. Profiling zCdh2 expression via antibody staining is essential to achieving better understanding of its role during zebrafish development. Generation of recombinant human antibodies to zCdh2 by phage display was used to identify monoclonal antibodies with reduced unspecific binding patterns when compared to available commercial antibodies. Specificity was profiled using flow cytometry of wild type, zCdh2-defective mutant and zCdh2-GFP zebrafish cells...
December 28, 2017: New Biotechnology
https://www.readbyqxmd.com/read/29285351/application-of-phage-display-for-the-development-of-a-novel-inhibitor-of-pla2-activity-in-western-cottonmouth-venom
#18
James K Titus, Matthew K Kay, Cdr Jacob J Glaser
Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A2 (PLA2) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized...
2017: Journal of Venom Research
https://www.readbyqxmd.com/read/29283291/structural-insights-into-humanization-of-anti-tissue-factor-antibody-10h10
#19
Alexey Teplyakov, Galina Obmolova, Thomas J Malia, Gopalan Raghunathan, Christian Martinez, Johan Fransson, Wilson Edwards, Judith Connor, Matthew Husovsky, Heena Beck, Ellen Chi, Sandra Fenton, Hong Zhou, Juan Carlos Almagro, Gary L Gilliland
Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody...
December 28, 2017: MAbs
https://www.readbyqxmd.com/read/29273737/hydrolytic-activity-determination-of-tail-tubular-protein-a-of-klebsiella-pneumoniae-bacteriophages-towards-saccharide-substrates
#20
Ewa Brzozowska, Anna Pyra, Krzysztof Pawlik, Monika Janik, Sabina Górska, Natalia Urbańska, Zuzanna Drulis-Kawa, Andrzej Gamian
In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A-TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K...
December 22, 2017: Scientific Reports
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