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Protein Engineering, Design & Selection: PEDS

Yu Gao, Jian-Jun Li, Lanyan Zheng, Yuguang Du
Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment...
November 7, 2017: Protein Engineering, Design & Selection: PEDS
B M Xavier, E Hildebrandt, F Jiang, H Ding, J C Kappes, I L Urbatsch
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
M Amitay, M Goldstein
DEEPSAM is a relatively new global optimization algorithm aimed to predict the structure of bio-molecules from sequence, without any additional preliminary assumption. It is an evolutionary algorithm whose mutation operators are built by hybridizing the diffusion equation method, molecular dynamics simulated annealing, and a quasi-Newton local minimization method. The goal of this study was to evaluate the structure prediction capabilities of DEEPSAM by running it upon NMR structures of linear peptides (10-20 residues)...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
J Wong, X Chen, K Truong
Since tobacco etch virus protease (TEVp) has a high specificity and efficiency in cleaving its target substrates, many groups have attempted to engineer conditional control of its activity. Temperature induction is widely used for modulating gene function because it has fast temporal response, good penetrability and applicability to many model organisms. Here, we engineered a temperature sensitive TEVp (tsTEVp) by using N-terminal truncations to TEVp that achieved efficient proteolysis on a timescale of 4 h after 30°C induction, while remaining relatively inactive at 37°C...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
Casey K Hua, Albert T Gacerez, Charles L Sentman, Margaret E Ackerman
As a stress-inducible natural killer (NK) cell ligand, B7H6 plays a role in innate tumor immunosurveillance and is a fairly tumor selective marker expressed on a variety of solid and hematologic cancer cells. Here, we describe the isolation and characterization of a new family of single chain fragment variable (scFv) molecules targeting the human B7H6 ligand. Through directed evolution of a yeast surface displayed non-immune human-derived scFv library, eight candidates comprising a single family of clones differing by up to four amino acid mutations and exhibiting nM avidities for soluble B7H6-Ig were isolated...
October 1, 2017: Protein Engineering, Design & Selection: PEDS
Paul W H I Parren, James S Huston
No abstract text is available yet for this article.
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Daniel Steiner, Frieder W Merz, Ivo Sonderegger, Maya Gulotti-Georgieva, Denis Villemagne, Douglas J Phillips, Patrik Forrer, Michael T Stumpp, Christof Zitt, H Kaspar Binz
A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
A Kuglstatter, M Stihle, C Neumann, C Müller, W Schaefer, C Klein, J Benz
An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Hok Seon Kim, Diana Ronai Dunshee, Angie Yee, Raymond K Tong, Ingrid Kim, Farzam Farahi, Jo-Anne Hongo, James A Ernst, Junichiro Sonoda, Christoph Spiess
Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Thorsten Gantke, Michael Weichel, Carmen Herbrecht, Uwe Reusch, Kristina Ellwanger, Ivica Fucek, Markus Eser, Thomas Müller, Remko Griep, Vera Molkenthin, Eugene A Zhukovsky, Martin Treder
Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
S Miersch, S Kuruganti, M R Walter, S S Sidhu
The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Maximilian Bönisch, Carolin Sellmann, Daniel Maresch, Claudia Halbig, Stefan Becker, Lars Toleikis, Björn Hock, Florian Rüker
Targeting two unique antigens with a single bispecific antibody is an attractive approach with potential broad therapeutic applicability. However, the production of heterodimeric bispecific antibodies (bsAbs) presents a challenge, requiring the co-expression and accurate pairing of two distinct heavy and light chain units. Several undesirable by-products can be formed in the production process, including heavy chain homodimers and non-cognate light chain pairings. Although additional downstream purification methods exist, they are often time consuming and restrict practical large-scale production...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Gordana Wozniak-Knopp, Gerhard Stadlmayr, Jan Walther Perthold, Katharina Stadlbauer, Maximilian Woisetschläger, Haijun Sun, Florian Rüker
Fc fragment with antigen-binding (Fcab) is a novel construct which can be selected to recognize specifically a wide variety of target proteins. We describe the selection and affinity maturation of Fcab clones targeting VEGF, an important pro-angiogenesis factor. To investigate the extent of engineering permissible to Fcabs we applied targeted mutagenesis to all three C-terminal loop structures and the C-terminus of the CH3 domain to isolate high-affinity binders by directed evolution and yeast display. The matured clone, CT6, binds to VEGF with low nanomolar affinity and inhibits VEGF-stimulated proliferation of human umbilical vein endothelial cells in vitro...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Xin Yu, Liang Qu, Darell D Bigner, Vidyalakshmi Chandramohan
Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human high-affinity CSPG4 antibodies or antibody fragments. To overcome this limitation, we performed affinity maturation on a novel human CSPG4 single-chain Fv fragment (scFv) using the random mutagenesis approach and screened for improved variants from a yeast display library using a modified whole-cell panning method followed by fluorescence-activated cell sorting...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
M Ultsch, A Braisted, H R Maun, C Eigenbrot
The well-studied B-domain from Staphylococcal protein A is a 59 amino acid three-helix bundle that binds the Fc portion of IgG with a dissociation constant of ~35 nM. The B-domain variant bearing a Gly to Ala mutation (=Z-domain) has been the subject of efforts to minimize a domain's size while retaining its function. We report X-ray crystallographic characterization of three steps in such a process using complexes with Fc: the full three-helix Z-domain, a 34 amino acid two-helix version called Z34C and a 13 amino acid single helix stabilized with an exo-helix tether, called LH1...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Masoumeh Nosrati, Sara Solbak, Olle Nordesjö, Mikael Nissbeck, Daniel F A R Dourado, Ken G Andersson, Mohammad Reza Housaindokht, John Löfblom, Anders Virtanen, U Helena Danielson, Samuel Coulbourn Flores
The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Ronit Rosenfeld, Ron Alcalay, Adva Mechaly, Gideon Lapidoth, Eyal Epstein, Chanoch Kronman, Sarel J Fleishman, Ohad Mazor
While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Lei Chen, Yuanyuan Duan, Lorenzo Benatuil, William B Stine
Clear and accurate understanding of diversity in antibody complementarity-determining regions (CDRs) is critical for antibody discovery and engineering. Previous observations of antibody CDR-H3 diversity were based on analyzing available antibody sequences in the public databases. The results may not accurately reflect that of natural antibody repertoire due to erroneous species annotation and the presence of man-made CDR loop diversity in public antibody sequence databases. In this study, in a precisely controlled germline context, we explored the relationship between amino acid composition and CDR-H3 length using 5518 unique productively rearranged human VH3-23*01 gene sequences...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Séverine Fagète, Ledicia Botas-Perez, Irène Rossito-Borlat, Kenneth Adea, Franck Gueneau, Ulla Ravn, François Rousseau, Marie Kosco-Vilbois, Nicolas Fischer, Oliver Hartley
Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly...
September 1, 2017: Protein Engineering, Design & Selection: PEDS
Jian Lu, Yuxia Dong, Emily C Ng, Daniel L Siehl
No abstract text is available yet for this article.
August 17, 2017: Protein Engineering, Design & Selection: PEDS
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