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Protein Engineering, Design & Selection: PEDS

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https://www.readbyqxmd.com/read/28201818/collective-repacking-reveals-that-the-structures-of-protein-cores-are-uniquely-specified-by-steric-repulsive-interactions
#1
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
#2
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/28201611/secretion-of-functional-formate-dehydrogenase-in-pichia-pastoris
#3
Michelle Takacs, Olga V Makhlynets, Patricia L Tolbert, Ivan V Korendovych
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
#4
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
#5
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
#6
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/28158843/probing-the-influence-of-non-covalent-contact-networks-identified-by-charge-density-analysis-on-the-oxidoreductase-bacc
#7
Kumar Perinbam, Hemalatha Balaram, Tayur N Guru Row, Balasubramanian Gopal
No abstract text is available yet for this article.
February 2, 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
#8
S Pundir, J Onwubiko, R Zaru, S Rosanoff, R Antunes, M Bingley, X Watkins, C O'Donovan, M J Martin
No abstract text is available yet for this article.
February 2, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28130327/insertion-of-inter-domain-linkers-improves-expression-and-bioactivity-of-zygote-arrest-zar-fusion-proteins
#9
Jonathan M Cook, Amanda Charlesworth
Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function...
January 26, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28130326/engineering-the-expression-of-an-anti-interleukin-13-antibody-through-rational-design-and-mutagenesis
#10
Bojana Popovic, Suzanne Gibson, Tarik Senussi, Sara Carmen, Sara Kidd, Tim Slidel, Ian Strickland, Xu Jianqing, Jennifer Spooner, Amanda Lewis, Nathan Hudson, Lorna Mackenzie, Jennifer Keen, Ben Kemp, Colin Hardman, Diane Hatton, Trevor Wilkinson, Tristan Vaughan, David Lowe
High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression...
January 26, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28100651/stabilization-of-bacillus-circulans-xylanase-by-combinatorial-insertional-fusion-to-a-thermophilic-host-protein
#11
Vandan Shah, Brennal Pierre, Tamari Kirtadze, Seung Shin, Jin Ryoun Kim
High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX)...
January 17, 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
#12
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...
January 10, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062648/dual-chemistry-catalyzed-by-human-acireductone-dioxygenase
#13
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 (Fe(2+) or Ni(2+)) 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...
January 5, 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
#14
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...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062646/generation-of-human-bispecific-common-light-chain-antibodies-by-combining-animal-immunization-and-yeast-display
#15
Simon Krah, Christian Schröter, Carla Eller, Laura Rhiel, Nicolas Rasche, Jan Beck, Carolin Sellmann, Ralf Günther, Lars Toleikis, Björn Hock, Harald Kolmar, Stefan Becker
Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062645/crystallographic-substrate-binding-studies-of-leishmania-mexicana-scp2-thiolase-type-2-unique-features-of-oxyanion-hole-1
#16
Rajesh K Harijan, Tiila-Riikka Kiema, Shahan M Syed, Imran Qadir, Muriel Mazet, Frédéric Bringaud, Paul A M Michels, Rik K Wierenga
Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( C: xS, N: EAF, G H: P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints C: xS, H: DCF and G H: P...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28062644/ligand-induced-conformational-changes-in-prolyl-oligopeptidase-a-kinetic-approach
#17
R Van Elzen, E Schoenmakers, I Brandt, P Van Der Veken, A M Lambeir
Most kinetic studies of prolyl oligopeptidase (PREP) were performed with the porcine enzyme using modified peptide substrates. Yet recent biophysical studies used the human homolog. Therefore, the aim of this study was to compare the kinetic behavior of human and porcine PREP, as well as to find a suitable method to study enzyme kinetics with an unmodified biological substrate. It was found that human PREP behaves identically to the porcine homolog, displaying a double bell-shaped pH profile and a pH-dependent solvent kinetic isotope effect of the kcat/Km, features that set it apart from the related exopeptidase dipeptidyl peptidase IV (DPP IV)...
January 5, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28044007/modification-of-the-peroxygenative-peroxidative-activity-ratio-in-the-unspecific-peroxygenase-from-agrocybe-aegerita-by-structure-guided-evolution
#18
Diana M Mate, Miguel A Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, Miguel Alcalde
Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values...
January 1, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28007937/directed-evolution-of-p450cin-for-mediated-electron-transfer
#19
Ketaki D Belsare, Thomas Horn, Anna Joëlle Ruff, Ronny Martinez, Anders Magnusson, Dirk Holtmann, Jens Schrader, Ulrich Schwaneberg
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions...
December 22, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27999093/a-robust-cosolvent-compatible-halohydrin-dehalogenase-by-computational-library-design
#20
Hesam Arabnejad, Marco Dal Lago, Peter A Jekel, Robert J Floor, Andy-Mark W H Thunnissen, Anke C Terwisscha van Scheltinga, Hein J Wijma, Dick B Janssen
To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective...
December 19, 2016: Protein Engineering, Design & Selection: PEDS
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