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Protein Engineering, Design & Selection: PEDS

Lilia A Rabia, Yulei Zhang, Seth D Ludwig, Mark C Julian, Peter M Tessier
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 (CDRs) is a strong predictor of antibody specificity...
February 16, 2019: Protein Engineering, Design & Selection: PEDS
Zachary T Britton, Timothy B London, Jeffrey Carrell, Bhupinder Dosanjh, Trevor Wilkinson, Michael A Bowen, Herren Wu, William F Dall'Acqua, Marcello Marelli, Yariv Mazor
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 form...
February 12, 2019: Protein Engineering, Design & Selection: PEDS
Makoto Nakabayashi, Saori Kamachi, Dominggus Malle, Toshiaki Yanamoto, Seiichiro Kishishita, Tatsuya Fujii, Hiroyuki Inoue, Kazuhiko Ishikawa
Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T...
January 31, 2019: Protein Engineering, Design & Selection: PEDS
Austin L Day, Per Greisen, Lindsey Doyle, Alberto Schena, Nephi Stella, Kai Johnsson, David Baker, Barry Stoddard
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) and was subjected to biochemical characterization, further selections for enhanced 25-D3 binding affinity and crystallographic analyses...
December 19, 2018: Protein Engineering, Design & Selection: PEDS
Wu-Mei Yuan, Rui Zhang, Qian Zhang, Fen-Lian Ma, Chao Wang, Ying-Zi Wang, Yan Zeng, Li-Shu Zheng
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 of rhIFN-λ1-CTPON by mass spectrometry and then measured its biological activities...
November 29, 2018: Protein Engineering, Design & Selection: PEDS
Malka Aker, Shirly Ohanona, Shira Fisher, Efrat Katsman, Shirit Dvorkin, Efrat Kopelowitz, Moshe Goldstein, Zohar Barnett-Itzhaki, Moshe Amitay
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, is a database web application for heterodimeric protein crystallographic complexes along with the crystallographic structures of each individual unbound protein. Direct access to crystallographic structures of protein complexes, along with provided annotations, can serve as starting point for constructing new experimental protein complexes sets of any type, for protein binding studies, and the development and evaluation of PPIs prediction methods...
November 17, 2018: Protein Engineering, Design & Selection: PEDS
Tian Jiang, P Douglas Renfrew, Kevin Drew, Noah Youngs, Glenn L Butterfoss, Richard Bonneau, Den Nis Shasha
A wide variety of protein and peptidomimetic design tasks require matching functional 3D motifs to potential oligomeric scaffolds. For example, during enzyme design, one aims to graft active-site patterns-typically consisting of 3-15 residues-onto new protein surfaces. Identifying protein scaffolds suitable for such active-site engraftment requires costly searches for protein folds that provide the correct side chain positioning to host the desired active site. Other examples of biodesign tasks that require similar fast exact geometric searches of potential side chain positioning include mimicking binding hotspots, design of metal binding clusters and the design of modular hydrogen binding networks for specificity...
November 8, 2018: Protein Engineering, Design & Selection: PEDS
Chang-Hao Wu, Cynthia A Ponir, Dominik K Haja, Michael W W Adams
The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme...
October 24, 2018: Protein Engineering, Design & Selection: PEDS
Ugur Pala, Berin Yelmazer, Meltem Çorbacioglu, Jouni Ruupunen, Jarkko Valjakka, Ossi Turunen, Baris Binay
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 (reduction) as a function of substrate concentration...
October 15, 2018: Protein Engineering, Design & Selection: PEDS
JdlM Castillo, S Caminata Landriel, M Sánchez Costa, O A Taboga, J Berenguer, A Hidalgo, S A Ferrarotti, H Costa
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 of engineered enzymes with improved yield and/or particular product specificity...
August 13, 2018: Protein Engineering, Design & Selection: PEDS
Candice Gautier, Louise Laursen, Per Jemth, Stefano Gianni
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 their actual existence in PDZ domains...
August 2, 2018: Protein Engineering, Design & Selection: PEDS
James S Huston
No abstract text is available yet for this article.
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Michael J Rudolph, David J Vance, Simon Kelow, Siva Krishna Angalakurthi, Sophie Nguyen, Simon A Davis, Yinghui Rong, C Russell Middaugh, David D Weis, Roland Dunbrack, John Karanicolas, Nicholas J Mantis
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 bound to RTA (2...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Sandeep Kumar, Kirk Roffi, Dheeraj S Tomar, David Cirelli, Nicholas Luksha, Danielle Meyer, Jeffrey Mitchell, Martin J Allen, Li Li
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) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
S-Y Lau, J W Siau, R M Sobota, C-I Wang, P Zhong, D P Lane, F J Ghadessy
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 optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Joerg Thomas Regula, Sabine Imhof-Jung, Michael Mølhøj, Joerg Benz, Andreas Ehler, Alexander Bujotzek, Wolfgang Schaefer, Christian Klein
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...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Laura S Mitchell, Lucy J Colwell
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 features of the paratopes of both data sets...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Raiji Kawade, Hiroki Akiba, Kevin Entzminger, Toshiaki Maruyama, C J Okumura, Kouhei Tsumoto
Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition was not altered by the mutation. The present result expands the understanding of how rabbit antibodies with kappa light chains gain affinity under characteristic mechanism to gain thermal stability, and would give suggestions for the methods to artificially stabilize antibody molecules...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
L Schwaigerlehner, M Pechlaner, P Mayrhofer, C Oostenbrink, R Kunert
Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
Xiufeng Wu, Richard Yuan, Michael Bacica, Stephen J Demarest
The clinical success of monoclonal antibodies to treat diseases across nearly every therapeutic area has spurred advances in bispecific antibody technology with the goal of cost-effectively combining various therapies or providing novel mechanisms for disease intervention. Many novel bispecific antibodies are now in clinical development or the late pre-clinical setting. A new horizon exists for novel molecular entities with the ability to bind three or more antigens. Here we describe the production and characterization of novel trispecific antibody-like proteins denoted 'OrthoTsAbs' that self-assemble through the application of engineered antibody domain interfaces...
July 1, 2018: Protein Engineering, Design & Selection: PEDS
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