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Affinity maturation

Magfur E Alam, Steven B Geng, Christian Bender, Seth D Ludwig, Lars Linden, Rene Hoet, Peter M Tessier
In vitro antibody discovery and/or affinity maturation are often performed using antibody fragments (Fabs), but most monovalent Fabs are reformatted as bivalent IgGs (monoclonal antibodies, mAbs) for therapeutic applications. One problem related to reformatting antibodies is that the bivalency of mAbs can lead to increased antibody self-association and poor biophysical properties (e.g., reduced antibody solubility and increased viscosity). Therefore, it is important to identify monovalent Fabs early in the discovery and/or optimization process that will display favorable biophysical properties when reformatted as bivalent mAbs...
November 20, 2017: Molecular Pharmaceutics
Anne Durand, Marie-Line Bourbon, Anne-Soisig Steunou, Bahia Khalfaoui-Hassani, Camille Legrand, Audrey Guitton, Chantal Astier, Soufian Ouchane
The cbb3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb3 are composed of four subunits: the catalytic CcoN subunit, the two cytochromes c subunits (CcoO and CcoP) involved in electron transfer and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly...
November 17, 2017: Journal of Biological Chemistry
Abram R Bernard, T Carson Jessop, Prashant Kumar, Nicholas E Dickenson
Type three secretion systems (T3SS) are specialized nano-machines that support infection by injecting bacterial proteins directly into host cells. The Shigella T3SS has uniquely evolved to sense environmental levels of the bile salt deoxycholate (DOC) and up-regulate virulence in response to DOC. In this study, we describe a rare i+5 hydrogen bonding secondary structure element (π-helix) within the type three secretion system tip protein IpaD that plays a critical role in DOC-enhanced virulence. Specifically, engineered mutations within the π-helix altered the pathogen's response to DOC with one mutant construct in particular exhibiting an unprecedented reduction in virulence following DOC exposure...
November 14, 2017: Biochemistry
Simon N Willis, Julie Tellier, Yang Liao, Stephanie Trezise, Amanda Light, Kristy O'Donnell, Lee Ann Garrett-Sinha, Wei Shi, David M Tarlinton, Stephen L Nutt
Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation...
November 10, 2017: Nature Communications
F Torabi, O A Bogle, J M Estanyol, R Oliva, D Miller
STUDY QUESTION: Are there novel hyaladherins in human sperm? SUMMARY ANSWER: Zona pellucida-binding protein 2 (ZPBP2), containing a Link-like hyaluronic acid (HA)-binding domain, and several other proteins containing BX7B motifs, such as ADAM32 and Midkine, may be novel hyaladherins with HA-binding properties. WHAT IS KNOWN ALREADY: HA-binding proteins (hyaladherins), which can bind HA surrounding the cumulus-oophorus complex, are distinct from hyases such as PH 20 (SPAM1) and are expressed by mature spermatozoa...
November 8, 2017: Molecular Human Reproduction
Owen M Pierce, Grant R McNair, Xu He, Hiroyuki Kajiura, Kazuhito Fujiyama, Allison R Kermode
Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-L-iduronidase (IDUA; EC enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity...
November 8, 2017: Plant Molecular Biology
Tobias Unkauf, Michael Hust, André Frenzel
Antibodies are the fastest growing class of pharmaceutical proteins and essential tools for research and diagnostics. Often antibodies do show a desirable specificity profile but lack sufficient affinity for the desired application. Here, we describe a method to increase the affinity of recombinant antibody fragments based on the construction of mutagenized phage display libraries.After the construction of a mutated antibody gene library by error-prone PCR, selection of high-affinity variants is either performed by panning in solution or on immobilized antigen with washing conditions optimized for off-rate-dependent selection...
2018: Methods in Molecular Biology
Julius Grzeschik, Doreen Könning, Steffen C Hinz, Simon Krah, Christian Schröter, Martin Empting, Harald Kolmar, Stefan Zielonka
Besides classical antibodies with the composition of heavy and light chains, sharks produce a unique heavy chain only isotype, termed Immunoglobulin New Antigen Receptor (IgNAR), in which antigen binding is solely mediated by a single domain, referred to as vNAR. Owing to their high affinity and specificity combined with their small size and high stability, vNAR domains emerged as promising target-binding scaffolds that can be tailor-made for biotechnological and biomedical applications. Herein, we describe protocols for the construction of semi-synthetic, CDR3-randomized vNAR libraries for the isolation of target-specific antibodies using yeast surface display or phage display as platform technology...
2018: Methods in Molecular Biology
Ramya Billur, David Ban, T Michael Sabo, Muriel Christine Maurer
Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge is lacking on changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I. Such changes are transient and failed to be captured by crystallography...
November 7, 2017: Biochemistry
A De Cola, M Franceschini, A Di Matteo, G Colotti, R Celani, E Clemente, R Ippoliti, A M Cimini, A C Dhez, B Vallée, F Raineri, I Cascone, D Destouches, V De Laurenzi, J Courty, L Federici
NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin...
October 27, 2017: Cancer Letters
John Steven, Mischa R Müller, Miguel F Carvalho, Obinna C Ubah, Marina Kovaleva, Gerard Donohoe, Thomas Baddeley, Dawn Cornock, Kenneth Saunders, Andrew J Porter, Caroline Jane Barelle
Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1...
2017: Frontiers in Immunology
Xiaoxiao Zhang, Peng Li, Jian Lin, Haochu Huang, Bin Yin, Yan Zeng
microRNAs (miRNAs) are first transcribed as long, primary transcripts, which are then processed by multiple enzymes and proteins to generate the single-stranded, approximately 22-nucleotide (nt)-long mature miRNAs. A critical step in animal miRNA biogenesis is the cleavage of primary miRNA transcripts (pri-miRNAs) to produce precursor miRNAs (pre-miRNAs) by the enzyme Drosha. How Drosha recognizes its substrates remains incompletely understood. In this study we constructed a series of human Drosha mutants and examined their enzymatic activities and interaction with RNAs...
November 3, 2017: Biochimica et Biophysica Acta
Jun Li, Wenyan Han, Kenneth A Pelkey, Jingjing Duan, Xia Mao, Ya-Xian Wang, Michael T Craig, Lijin Dong, Ronald S Petralia, Chris J McBain, Wei Lu
In the brain, many types of interneurons make functionally diverse inhibitory synapses onto principal neurons. Although numerous molecules have been identified to function in inhibitory synapse development, it remains unknown whether there is a unifying mechanism for development of diverse inhibitory synapses. Here we report a general molecular mechanism underlying hippocampal inhibitory synapse development. In developing neurons, the establishment of GABAergic transmission depends on Neuroligin 2 (NL2), a synaptic cell adhesion molecule (CAM)...
November 15, 2017: Neuron
Thomas M Schmitt, David H Aggen, Kumiko Ishida-Tsubota, Sebastian Ochsenreither, David M Kranz, Philip D Greenberg
Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs...
November 6, 2017: Nature Biotechnology
Peng Xu, Peter A Andreasen, Mingdong Huang
This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively...
2017: International Journal of Biological Sciences
Fei Li, Yi Huang, You-Ying Huang, Yan-Song Kuang, Yong-Jian Wei, Li Xiang, Xing-Ju Zhang, Zheng-Cai Jia, Shan Jiang, Jing-Yi Li, Ying Wan
B cells play a critical role in immune responses both in physiological and pathological conditions, and microRNAs have been shown to play important roles in regulating B cell proliferation and function. MiR-146a has been shown to modulate T cell immunity, but its function in regulating B cell response remains partially understood. Our previous studies indicated that germinal center (GC) B cells are significantly expanded in miR-146a-overexpressing (TG) mice. In this study, we further characterized the roles of miR-146a in regulating humoral immune responses to specific antigens...
November 1, 2017: Molecular Immunology
Wiebke Manuela Schulze, Stephen Cusack
Pol II transcribes diverse classes of RNAs that need to be directed into the appropriate nuclear maturation pathway. All nascent Pol II transcripts are 5'-capped and the cap is immediately sequestered by the nuclear cap-binding complex (CBC). Mutually exclusive interactions of CBC with different partner proteins have been implicated in transcript fate determination. Here, we characterise the direct interactions between CBC and NELF-E, a subunit of the negative elongation factor complex, ARS2 and PHAX. Our biochemical and crystal structure results show that the homologous C-terminal peptides of NELF-E and ARS2 bind identically to CBC and in each case the affinity is enhanced when CBC is bound to a cap analogue...
November 3, 2017: Nature Communications
Laura F Corns, Jing-Yi Jeng, Guy P Richardson, Corné J Kros, Walter Marcotti
The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (~>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (~P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel...
2017: Frontiers in Molecular Neuroscience
Ing-Chien Chen, Yi-Kai Chiu, Chung-Ming Yu, Cheng-Chung Lee, Chao-Ping Tung, Yueh-Liang Tsou, Yi-Jen Huang, Chia-Lung Lin, Hong-Sen Chen, Andrew H-J Wang, An-Suei Yang
Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies...
October 31, 2017: Scientific Reports
Kamini Gautam, Dasvit Shetty, Vikas D Trivedi, Madhushri Varunjikar, Prashant S Phale
Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA encoding CH in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4 to 6-fold higher in the periplasm as compared to the cytoplasm. The recombinant CH (rCH) showed 4-fold higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of Tmd+Sp resulted in expression of the inactive protein...
October 27, 2017: Applied and Environmental Microbiology
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