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Protein Engineering, Design & Selection: PEDS

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https://www.readbyqxmd.com/read/28444399/development-of-a-multipurpose-scaffold-for-the-display-of-peptide-loops
#1
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
#2
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
#3
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
#4
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
#5
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...
March 9, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338893/site-directed-mutant-libraries-for-isolating-minimal-mutations-yielding-functional-changes
#6
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...
March 9, 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
#7
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...
February 22, 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
#8
J C Gaines, A Virrueta, D A Buch, S J Fleishman, C S O'Hern, L Regan
No abstract text is available yet for this article.
February 15, 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
#9
Kusum Solanki, Walaa Abdallah, Scott Banta
No abstract text is available yet for this article.
February 15, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28180900/engineering-potent-long-acting-variants-of-the-wnt-inhibitor-dkk2
#10
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
No abstract text is available yet for this article.
February 9, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28160000/engineering-carboxypeptidase-g2-circular-permutations-for-the-design-of-an-autoinhibited-enzyme
#11
Brahm J Yachnin, Sagar D Khare
No abstract text is available yet for this article.
February 4, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28159998/overcoming-an-optimization-plateau-in-the-directed-evolution-of-highly-efficient-nerve-agent-bioscavengers
#12
Moshe Goldsmith, Nidhi Aggarwal, Yacov Ashani, Halim Jubran, Per Jr Greisen, Sergey Ovchinnikov, Haim Leader, David Baker, Joel L Sussman, Adi Goldenzweig, Sarel J Fleishman, Dan S Tawfik
No abstract text is available yet for this article.
February 4, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338942/structure-and-mechanism-of-benzaldehyde-dehydrogenase-from-pseudomonas-putida-atcc-12633-a-member-of-the-class-3-aldehyde-dehydrogenase-superfamily
#13
Megan P D Zahniser, Shreenath Prasad, Malea M Kneen, Cheryl A Kreinbring, Gregory A Petsko, Dagmar Ringe, Michael J McLeish
Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28338744/understanding-the-molecular-mechanism-of-substrate-channeling-and-domain-communication-in-protozoal-bifunctional-ts-dhfr
#14
Karen S Anderson
Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg) and Cryptosporidium hominis (Ch), contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the two sequential catalytic activities contained on a single polypeptide chain...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28201611/secretion-of-functional-formate-dehydrogenase-in-pichia-pastoris
#15
Michelle Takacs, Olga V Makhlynets, Patricia L Tolbert, Ivan V Korendovych
Biofuels are an important tool for the reduction of carbon dioxide and other greenhouse emissions. NAD+-dependent formate dehydrogenase has been previously shown to be capable of the electrochemical reduction of carbon dioxide into formate, which can be ultimately converted to methanol. We established that a functional enzyme, tagged for immobilization, could be continuously secreted by Pichia pastoris. The protein can be easily separated from the growth media and its activity remains constant over an extended period of time...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28158843/probing-the-influence-of-non-covalent-contact-networks-identified-by-charge-density-analysis-on-the-oxidoreductase-bacc
#16
Kumar Perinbam, Hemalatha Balaram, Tayur N Guru Row, Balasubramanian Gopal
Bacillus subtilis BacC is an oxidoreductase involved in the biosynthesis of the potent antibiotic bacilysin. The crystal structure of BacC was determined at 1.19 Å resolution. An experimental charge density approach was used to calculate non-covalent interactions within the monomer and across the dimeric interface of BacC. This interaction network, in turn, enabled an analysis of non-covalently connected paths that span the protein structure. One of the pathways of non-covalent interactions was examined by mutational analysis...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28158609/an-update-on-the-enzyme-portal-an-integrative-approach-for-exploring-enzyme-knowledge
#17
S Pundir, J Onwubiko, R Zaru, S Rosanoff, R Antunes, M Bingley, X Watkins, C O'Donovan, M J Martin
Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al. , (2013), BMC Bioinformatics , (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28073960/ligand-characterization-of-cyp4b1-isoforms-modified-for-high-level-expression-in-escherichia-coli-and-hepg2-cells
#18
Katharina Roellecke, Vera D Jäger, Veselin H Gyurov, John P Kowalski, Stephanie Mielke, Allan E Rettie, Helmut Hanenberg, Constanze Wiek, Marco Girhard
Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062648/dual-chemistry-catalyzed-by-human-acireductone-dioxygenase
#19
Aditi R Deshpande, Thomas C Pochapsky, Gregory A Petsko, Dagmar Ringe
Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062647/on-the-effect-of-alkaline-ph-and-cofactor-availability-in-the-conformational-and-oligomeric-state-of-escherichia-coli-glutamate-decarboxylase
#20
F Giovannercole, C Mérigoux, C Zamparelli, D Verzili, G Grassini, M Buckle, P Vachette, D De Biase
Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence...
March 1, 2017: Protein Engineering, Design & Selection: PEDS
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