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Protein Engineering, Design & Selection: PEDS

Angelo Toto, Annalisa Mattei, Per Jemth, Stefano Gianni
The PDZ domain is one of the most common protein-protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL...
October 19, 2016: Protein Engineering, Design & Selection: PEDS
Felix Bäuerle, Agnes Zotter, Gideon Schreiber
With computer-based data-fitting methods becoming a standard tool in biochemistry, progress curve analysis of enzyme kinetics is a feasible, yet seldom used tool. Here we present a versatile Matlab-based tool (PCAT) to analyze catalysis progress curves with three complementary model approaches. The first two models are based on the known closed-form solution for this problem: the first describes the required Lambert W function with an analytical approximation and the second provides a numerical solution of the Lambert W function...
October 15, 2016: Protein Engineering, Design & Selection: PEDS
Shalom D Goldberg, Rosa M F Cardoso, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven A Jacobs, Vadim Dudkin, Gary Gilliland, Karyn T O'Neil
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
Jan Neumann, Kay-Eberhard Gottschalk
Integrins are the major transmembrane cellular adhesion receptors. Talin binds to integrins with its head domain and links them to the actin cytoskeleton with its rod domain, acting as the force linkage between the extracellular matrix and the cytoskeleton. It is unknown how forces in different directions affect the integrin-talin complex. We show that small forces applied to the integrin-talin complex breaks a salt bridge between the integrins α- and β-subunit, unlocking the integrin from its resting state...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
Shangfei Zhang, Limei Zhu, Jie Yu, Jun Xu, Bin Gao, Changlin Zhou, Shunyi Zhu
Grafting of exogenous bioactive sites or functional motifs onto structurally stable scaffolds to gain new functions represents an important research direction in protein engineering. Some engineered proteins have been developed into therapeutic drugs. MeuNaTxα-3 (abbreviated as MT-3) is a newly characterized scorpion sodium channel toxin-like peptide isolated from the venom of the scorpion Mesobuthus eupeus, which contains a rigid scaffold highly similar to classical scorpion sodium channel toxins and an extension of eight amino acids in its J-loop region...
September 26, 2016: Protein Engineering, Design & Selection: PEDS
Tadas Povilaitis, Gediminas Alzbutas, Rasa Sukackaite, Juozas Siurkus, Remigijus Skirgaila
Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification...
September 26, 2016: Protein Engineering, Design & Selection: PEDS
Byron Carpenter, Christopher G Tate
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging...
September 26, 2016: Protein Engineering, Design & Selection: PEDS
Timothy P Riley, Cory M Ayres, Lance M Hellman, Nishant K Singh, Michael Cosiano, Jennifer M Cimons, Michael J Anderson, Kurt H Piepenbrink, Brian G Pierce, Zhiping Weng, Brian M Baker
T-cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for cancer where they are the key components of new cellular therapies as well as soluble biologics. Many studies have generated high affinity TCRs in order to enhance sensitivity. Recent outcomes, however, have suggested that fine manipulation of TCR binding, with an emphasis on specificity may be more valuable than large affinity increments. Structure-guided design is ideally suited for this role, and here we studied the generality of structure-guided design as applied to TCRs...
September 13, 2016: Protein Engineering, Design & Selection: PEDS
Margo Diricks, Alexander Gutmann, Simon Debacker, Griet Dewitte, Bernd Nidetzky, Tom Desmet
Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower Km value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP...
September 1, 2016: Protein Engineering, Design & Selection: PEDS
Li-Jen Hsu, Ning-Shian Hsu, Yung-Lin Wang, Chang-Jer Wu, Tsung-Lin Li
In the development of new functionalities of transketolase for the industrial strain Pichia stipitis (TKps) the structural information of TKps would allow us to gain insight into the enzyme's reaction mechanisms, substrates selectivity and reaction directionality to help reach the goal. We here report seven TKps crystal structures of wild type (WT) and mutants in complex with various physiological ligands. These complexes were refined to resolutions at 1.6-1.03 Å. Both biochemical and mutagenic analyses concluded that residues His27, His66, His100, His261, His478, Asp473, Arg356 and Arg525 play important roles in coenzyme binding and substrates recognition...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Cheng-Yi Chiang, Cheng-Yung Lin, Yen-Ting Chen, Huai-Jen Tsai
Chromoproteins, especially far-red fluorescent proteins with long stokes shift, are good sources for engineering biological research tools. However, chromoproteins have not been used for developing fluorescent proteins with short emission wavelength. Therefore, we herein report the development of a blue fluorescent protein, termed shBFP, which is derived from a purple chromoprotein isolated from the sea anemone Stichodacyla haddoni (shCP) after shCP was simultaneously mutated on E63L and Y64L. The shBFP chromophore is composed of Leu-Leu-Gly, which introduced a maximum excitation and emission wavelength at 401 nm and 458 nm, respectively, and a quantum yield of 0...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Benjamin T Porebski, Paul J Conroy, Nyssa Drinkwater, Peter Schofield, Rodrigo Vazquez-Lombardi, Morag R Hunter, David E Hoke, Daniel Christ, Sheena McGowan, Ashley M Buckle
The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Alexandra Lerchner, Marina Daake, Alexander Jarasch, Arne Skerra
To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT(L417M), as proven by SDS-PAGE and size exclusion chromatography...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Shuo Yao, Darren J Hart, Yingfeng An
As one of the simplest and most efficient cloning methods, T-vector-based TA cloning has been widely used for cloning of single genes and construction of DNA libraries. This approach is especially suitable for high-throughput cloning of diverse DNA fragments since inserts can be cloned without knowledge of their sequence; it is therefore an ideal tool for high-throughput analysis of protein structure and function. Although most of the currently available T-vectors can only be used for cloning purposes, some novel variants with improved functions have be developed...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Tim D Jones, Arron R Hearn, Robert G E Holgate, Dorota Kozub, Mark H Fogg, Francis J Carr, Matthew P Baker, Javier Lacadena, Kurt R Gehlsen
Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids...
August 29, 2016: Protein Engineering, Design & Selection: PEDS
Hiroshi Nishigami, Narutoshi Kamiya, Haruki Nakamura
The antigen-binding site of antibodies, also known as complementarity-determining region (CDR), has hypervariable sequence properties. In particular, the third CDR loop of the heavy chain, CDR-H3, has such variability in its sequence, length, and conformation that ordinary modeling techniques cannot build a high-quality structure. At Stage 2 of the Second Antibody Modeling Assessment (AMA-II) held in 2013, the model structures of the CDR-H3 loops were submitted by the seven modelers and were critically assessed...
August 11, 2016: Protein Engineering, Design & Selection: PEDS
James A Van Deventer, Doris N Le, Jessie Zhao, Haixing P Kehoe, Ryan L Kelly
The combination of protein display technologies and noncanonical amino acids (ncAAs) offers unprecedented opportunities for the high throughput discovery and characterization of molecules suitable for addressing fundamental and applied problems in biological systems. Here we demonstrate that ncAA-compatible yeast display facilitates evaluations of conjugation chemistry and stability that would be challenging or impossible to perform with existing mRNA, phage, or E. coli platforms. Our approach enables site-specific introduction of ncAAs into displayed proteins, robust modification at azide-containing residues, and quantitative evaluation of conjugates directly on the yeast surface...
August 11, 2016: Protein Engineering, Design & Selection: PEDS
James S Huston
No abstract text is available yet for this article.
October 2016: Protein Engineering, Design & Selection: PEDS
Kevin A Henry, Greg Hussack, Cathy Collins, John C Zwaagstra, Jamshid Tanha, C Roger MacKenzie
The epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-β3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VhH library against TGF-β3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-β3 binding and neutralized TGF-β3 in in vitro cellular assays...
October 2016: Protein Engineering, Design & Selection: PEDS
Anna Säll, Maria Walle, Christer Wingren, Susanne Müller, Tomas Nyman, Andrea Vala, Mats Ohlin, Carl A K Borrebaeck, Helena Persson
Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated...
October 2016: Protein Engineering, Design & Selection: PEDS
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