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Sources of Essential Oils
Published in K. Hüsnü Can Başer, Gerhard Buchbauer, Handbook of Essential Oils, 2020
Chlodwig Franz, Johannes Novak
Protein engineering is the application of scientific methods (mathematical and laboratory methods) to develop useful or valuable proteins. There are two general strategies for protein engineering, random mutagenesis and rational design. In rational design, detailed knowledge of the structure and function of the protein is necessary to make desired changes by site-directed mutagenesis, a technique already well developed. An impressive example of the rational design of monoterpene synthases was given by Kampranis et al. (2007) who converted a 1,8-cineole synthase from S. fruticosa into a synthase producing sabinene, the precursor of α- and β-thujones with a minimum number of substitutions. They went also a step further and converted this monoterpene synthase into a sesquiterpene synthase by substituting a single amino acid that enlarged the cavity of the active site enough to accommodate the larger precursor of the sesquiterpenes, farnesyl pyrophosphate.
Conversion of Natural Products from Renewable Resources in Pharmaceuticals by Cytochromes P450
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Giovanna Di Nardo, Gianfranco Gilardi
The crystal structures of cytochromes P450 show that they share a similar and conserved molecular scaffold with defined regions supporting their variability in the substrates and reaction catalyzed. Indeed, they are very good candidates for protein engineering targeting the variable regions with the aim of changing the substrate selectivity as well as regio- and steroselectivity (Whitehouse et al., 2012; McIntosh et al., 2014; Girvan and Munro, 2016). Site directed mutagenesis and directed evolution approaches have been successfully applied to generate mutants with improved features for biocatalytic applications in the pharmaceutical field (Kumar, 2010; Di Nardo and Gilardi, 2012; Gillam and Hayes, 2013; Caswell et al., 2013). Moreover, cytochromes P450 are quite versatile in the use of a redox partner for catalysis. Thus, when the physiological redox partner is not available, it can be substituted by a nonphysiological one (O’Keefe et al., 1991; Chun et al., 2006; Urlacher and Girhard, 2012; Ringle et al., 2013; Milhim et al., 2016) and, when the redox partner is a different protein, its fusion at genic level with the cytochrome P450 can produce self-sufficient enzymes, retaining the same catalytic efficiency as the separate system (Sadeghi and Gilardi, 2013; Kang et al., 2014; Bakkes et al., 2017; Degregorio et al., 2017; Castrignanò et al., 2018). Interestingly, it has been observed that the use of different redox partner with the same P450 enzyme can result in different reactions and products from the same substrate (Zhang et al., 2014).
Structure and function of Human CYP2D6
Published in Shufeng Zhou, Cytochrome P450 2D6, 2018
Ellis et al. (1996) developed a homology model of the CYP2D6 active site on the basis of the bacterial CYP102 structure, which is similar to that derived by Lewis (1995) with a minor modification in the β1-4 region, namely, movement of the sequence by one residue, such that positions Val374 and His376 of CYP2D6 are in alignment with Phe331 and Leu333 of CYP102, respectively. In this homology model, the active site consisted of a cavity bordered by hydrophobic residues, and important active-site residues included Thr309 and Thr312 of the oxygen-binding site, with Asp301, Ser304, and Ala305 lying in the I helix and Pro371, Gly373, and Val374 of the β1-4-sheet region (Ellis et al. 1996). The active-site area is further defined by a lipophilic pocket bordered by Val480 and Phe481 of the loop and β6-2 region. This model identifies Asp301 as the critical substrate-contact residue involved in the proposed electrostatic interaction between the basic nitrogen of typical substrates of CYP2D6 and a negatively charged site in the active site. This is consistent with the data from site-directed mutagenesis studies (Ellis et al. 1995).
Production, characterization, and in vivo half-life extension of polymeric IgA molecules in mice
Published in mAbs, 2019
T. Noelle Lombana, Sharmila Rajan, Julie A. Zorn, Danielle Mandikian, Eugene C. Chen, Alberto Estevez, Victor Yip, Daniel D. Bravo, Wilson Phung, Farzam Farahi, Sharon Viajar, Sophia Lee, Avinash Gill, Wendy Sandoval, Jianyong Wang, Claudio Ciferri, C. Andrew Boswell, Marissa L. Matsumoto, Christoph Spiess
Antibody variable domain sequences used include a humanized anti-human HER2 antibody54 and a murine anti-murine IL-13 antibody (Genentech). Protein sequences of human IgA constant heavy chains IgA1, IgA2m1, and IgA2m2, other IgA species, and human J chain were obtained from UniProt (www.uniprot.org) or NCBI (www.ncbi.nlm.nih.gov/protein). P221R is a mutation in IgA2m1 that stabilizes the light chain-heavy chain disulfide as previously reported.24 Genes encoding a fusion of the antibody variable domains to the human light chain and human IgA1, IgA2m1, and IgA2m2 heavy chain constant domains were synthesized and cloned into the mammalian pRK vector.55 Site-directed mutagenesis was used to introduce point mutations. All plasmids were sequence-verified. Sequence alignments were done using GSeqWeb (Genentech) and Excel (Microsoft).
Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody
Published in mAbs, 2018
Dana M. Lord, Julie J. Bird, Denise M. Honey, Annie Best, Anna Park, Ronnie R. Wei, Huawei Qiu
Site-directed mutagenesis was performed aiming to introduce flexibility into the Fab versions. For the first round of Fab mutations, additional amino acids were inserted in the light chain (LC) elbow region. More specifically, mutants were designed to add one glycine (L1), two glycines (L2), two glycines and one serine (L3), three glycines and one serine (L4), and four glycines and one serine (L5) sequences into the wild type (WT) light chain elbow region (Table 1). The light chain mutants bound to TGFβ1 with a significantly increased affinity compared to the WT CAT192 Fab (Table 2). These results show a step-wise improvement in TGFβ1 binding with the insertion of each additional residue into the elbow region of the Fab. None of the CAT192 LC Fab mutants bound to TGFβ2 or TGFβ3 (result not shown), demonstrating that the mutants retained isoform-selectivity.
Clinical and virological implications of A1846T and C1913A/G mutations of hepatitis B virus genome in severe liver diseases
Published in Scandinavian Journal of Gastroenterology, 2018
Hong Zang, Zhihui Xu, Yan Liu, Xiaodong Li, Yihui Rong, Ling Jiang, Shaoli You, Jinhua Hu, Jun Zhao, Dongping Xu, Shaojie Xin
Wild-type counterparts were generated from clinically mutated viral strains by using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and standard cloning techniques according to Günther’s method [27] with some modification as we described previously [29–31]. Briefly, HBV DNA of genotype C (a type mainly distributed in Asians) extracted from patients with mutations of A1762T + G1764A, A1846T, C1913A, C1913G, A1846T with C1913A (A1846T + C1913A), and A1846T with C1913G (A1846T + C1913G), respectively were used as templates for the construction of 1.0-unit HBV genomes. The primers used for site-directed mutagenesis were 5′-AGATTAGGTTAAAGGTCTTTGTACTAGGAGG-3′ (sense, nt 1750–1772) and 5′-CCTCCTAGTACA AAGACC TTTAACCTAATCT-3′ (antisense, nt 1750–1772). Site-directed mutagenesis was confirmed by sequencing analysis.