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Protein Engineering, Design & Selection: PEDS

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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
#1
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 9, 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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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/28338744/understanding-the-molecular-mechanism-of-substrate-channeling-and-domain-communication-in-protozoal-bifunctional-ts-dhfr
#9
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 majorg(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...
February 4, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28160000/engineering-carboxypeptidase-g2-circular-permutations-for-the-design-of-an-autoinhibited-enzyme
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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