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Protein Engineering, Design & Selection: PEDS

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https://www.readbyqxmd.com/read/28633311/cofactor-specificity-switch-in-shikimate-dehydrogenase-by-rational-design-and-consensus-engineering
#1
Fernando García-Guevara, Iris Bravo, Claudia Martínez-Anaya, Lorenzo Segovia
Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design...
June 13, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28541524/metal-bound-clamp-tag-inhibits-proteolytic-cleavage
#2
Michaela L McNiff, Jennifer S Chadwick
Biologics can be an improvement to small molecule drugs, providing high specificity for an identified target, lowering toxicity and limiting side effects. To achieve effective delivery, the biologic must have sufficient time to reach the target tissue. A prolonged half-life in the circulating environment is desired, but often serum stability is limited by proteases. Proteolysis in the serum causes degradation and inactivation as the biologic is fragmented and more rapidly cleared from the body. To improve the circulating half-life, large, hydrophilic polymers may be conjugated or stable fusion tags may be engineered to increase the effective size of the peptide and to hinder degradation by proteases...
May 25, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28482039/engineered-bacillus-pumilus-laccase-like-multi-copper-oxidase-for-enhanced-oxidation-of-the-lignin-model-compound-guaiacol
#3
Julian Ihssen, Dagmara Jankowska, Thomas Ramsauer, Renate Reiss, Ronny Luchsinger, Luzia Wiesli, Mark Schubert, Linda Thöny-Meyer, Greta Faccio
Laccases and laccase-like multi-copper oxidases (LMCOs) are versatile and robust biocatalysts applied in a variety of oxidative processes, and various studies have attempted to improve their catalytic activity. Here we report the engineering of a bacterial LMCO for enhanced oxidation of the lignin-related compound guaiacol by a combination of structure-guided mutagenesis and DNA shuffling. Mutant L9 showed a 1.39 mM Km for guaiacol and a 2.5-fold increase in turnover rate (kcat/Km = 2.85·104 M-1s-1).
May 8, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28475759/newprot-a-protein-engineering-portal
#4
Andreas Schwarte, Maika Genz, Lilly Skalden, Alberto Nobili, Clare Vickers, Okke Melse, Remko Kuipers, Henk-Jan Joosten, Jan Stourac, Jaroslav Bendl, Jon Black, Peter Haase, Coos Baakman, Jiri Damborsky, Uwe Bornscheuer, Gert Vriend, Hanka Venselaar
The NewProt protein engineering portal is a one-stop-shop for in silico protein engineering. It gives access to a large number of servers that compute a wide variety of protein structure characteristics supporting work on the modification of proteins through the introduction of (multiple) point mutations. The results can be inspected through multiple visualizers. The HOPE software is included to indicate mutations with possible undesired side effects. The Hotspot Wizard software is embedded for the design of mutations that modify a proteins' activity, specificity, or stability...
May 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28475752/3-2-1-structural-insights-from-stepwise-shrinkage-of-a-three-helix-fc-binding-domain-to-a-single-helix
#5
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...
May 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28475686/quercitrin-and-quercetin-3-%C3%AE-d-glucoside-as-chemical-chaperones-for-the-a4v-sod1-als-causing-mutant
#6
Philbert Ip, Priya Roy Sharda, Anna Cunningham, Sumon Chakrabartty, Vijay Pande, Avijit Chakrabartty
In many cases of familial amyotrophic lateral sclerosis (ALS), mutant forms of the Cu,Zn superoxide dismutase protein (SOD1) misfold and aggregate in motor neurons. Monomers of the normally homodimeric SOD1 have been found in patient tissue, presymptomatic mouse models of ALS, and in vitro misfolding assays which suggests that monomerization might be an early step in the pathological SOD1 misfolding pathway. In this study, we targeted the dimer interface with small molecules that might act as chemical chaperones to stabilize the native dimer and prevent downstream misfolding and aggregation...
May 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28472513/insights-from-engineering-the-affibody-fc-interaction-with-a-computational-experimental-method
#7
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...
May 3, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28472478/improved-antibody-based-ricin-neutralization-by-affinity-maturation-is-correlated-with-slower-off-rate-values
#8
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...
May 3, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28472386/analysis-of-5518-unique-productively-rearranged-human-vh3-23-01-gene-sequences-reveals-cdr-h3-length-dependent-usage-of-the-ighd2-gene-family
#9
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...
May 3, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28453776/modulating-and-evaluating-receptor-promiscuity-through-directed-evolution-and-modeling
#10
Sarah C Stainbrook, Jessica S Yu, Michael P Reddick, Neda Bagheri, Keith E J Tyo
The promiscuity of G-protein-coupled receptors (GPCRs) has broad implications in disease, pharmacology and biosensing. Promiscuity is a particularly crucial consideration for protein engineering, where the ability to modulate and model promiscuity is essential for developing desirable proteins. Here, we present methodologies for (i) modifying GPCR promiscuity using directed evolution and (ii) predicting receptor response and identifying important peptide features using quantitative structure-activity relationship models and grouping-exhaustive feature selection...
April 27, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28444399/development-of-a-multipurpose-scaffold-for-the-display-of-peptide-loops
#11
Maxim Rossmann, Sandra J Greive, Tommaso Moschetti, Michael Dinan, Marko Hyvönen
Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization...
April 24, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28444392/the-embo-biocatalysis-conference-the-biochemistry-and-chemistry-of-biocatalysis-from-understanding-to-design
#12
Rik K Wierenga, Dagmar Ringe
No abstract text is available yet for this article.
April 24, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28444391/dual-display-phage-selection-driven-by-co-engagement-of-two-targets-by-two-different-antibody-fragments
#13
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...
April 24, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28431161/engineering-a-high-affinity-peptide-binding-site-into-the-anti-cea-mab-m5a
#14
Cindy Zer, Kendra N Avery, Kassondra Meyer, Leah Goodstein, Krzysztof P Bzymek, Gagandeep Singh, John C Williams
We have previously identified a cyclic peptide called meditope which binds to the central cavity of the Fab portion of cetuximab and shown that this peptide binding site can be grafted, or 'meditope-enabled', onto trastuzumab. This peptide has been shown to act as a hitch for the non-covalent attachment of imaging agents to meditope-enabled antibodies. Herein, we explore the process of grafting this peptide binding site onto M5A, an anti-CEA antibody in clinical trials for cancer diagnostics. In order to explore the contributions of the amino acids, we sequentially introduced pairs of amino acid substitutions into the Fab and then we reverse-substituted key residues in the presence of the other substitutions...
April 19, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338903/disulfide-bridges-as-essential-elements-for-the-thermostability-of-lytic-polysaccharide-monooxygenase-lpmo10c-from-streptomyces-coelicolor
#15
Magali Tanghe, Barbara Danneels, Matthias Last, Koen Beerens, Ingeborg Stals, Tom Desmet
Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338893/site-directed-mutant-libraries-for-isolating-minimal-mutations-yielding-functional-changes
#16
Dong Hee Chung, Sarah C Potter, Ammon C Tanomrat, Krishnakumar M Ravikumar, Michael D Toney
Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338799/novel-form-of-the-michaelis-menten-equation-that-enables-accurate-estimation-of-kcat-km-ki-with-just-two-rate-measurements-utility-in-directed-evolution
#17
Jian Lu, Yuxia Dong, Emily C Ng, Daniel L Siehl
One of applications of directed evolution is to desensitize an enzyme to an inhibitor. kcat,1/KM and KI are three dimensions that when multiplied measure an enzyme's intrinsic capacity for catalysis in the presence of an inhibitor. The ideal values for the individual dimensions depend on substrate and inhibitor concentrations under the conditions of the application. When attempting to optimize those values by directed evolution, (kcat/KM)*KI can be an informative parameter for evaluating libraries of variants, but throughput is limited...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28201818/collective-repacking-reveals-that-the-structures-of-protein-cores-are-uniquely-specified-by-steric-repulsive-interactions
#18
J C Gaines, A Virrueta, D A Buch, S J Fleishman, C S O'Hern, L Regan
Protein core repacking is a standard test of protein modeling software. A recent study of six different modeling software packages showed that they are more successful at predicting side chain conformations of core compared to surface residues. All the modeling software tested have multicomponent energy functions, typically including contributions from solvation, electrostatics, hydrogen bonding and Lennard-Jones interactions in addition to statistical terms based on observed protein structures. We investigated to what extent a simplified energy function that includes only stereochemical constraints and repulsive hard-sphere interactions can correctly repack protein cores...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28201792/engineering-the-cofactor-specificity-of-an-alcohol-dehydrogenase-via-single-mutations-or-insertions-distal-to-the-2-phosphate-group-of-nadp-h
#19
Kusum Solanki, Walaa Abdallah, Scott Banta
There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2'-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a 'loop-grafting' approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28180900/engineering-potent-long-acting-variants-of-the-wnt-inhibitor-dkk2
#20
Richelle Sopko, Joshua W Mugford, Andreas Lehmann, Renée I Shapiro, Mia Rushe, Abhishek Kulkarni, Joseph Worrall, Joseph Amatucci, Dingyi Wen, Nels E Pederson, Brenda K Minesinger, Joseph W Arndt, Blake Pepinsky
Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide...
May 1, 2017: Protein Engineering, Design & Selection: PEDS
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