title: Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris.
abstract: The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.
authors: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
authors: T
authors: T
authors: Kenzom
authors: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India. Electronic address: preetisrivastava@hotmail.com.
authors: P
authors: P
authors: Srivastava
authors: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
authors: S
authors: S
authors: Mishra
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D052160:Benzothiazoles / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D002999:Clone Cells; D003124:Colorimetry; D004396:Coloring Agents / Q000378:metabolism; D004790:Enzyme Induction; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D007700:Kinetics; D042845:Laccase / Q000235:genetics / Q000378:metabolism*; D000432:Methanol / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D013451:Sulfonic Acids / Q000378:metabolism; D000099188:Alternative Oxidase
chemical_list: D000429:Alcohol Oxidoreductases; D052160:Benzothiazoles; D001761:Bleomycin; D004396:Coloring Agents; D005656:Fungal Proteins; D042845:Laccase; D000432:Methanol; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D000099188:Alternative Oxidase; C105427:Zeocin; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C019643:alcohol oxidase
keywords: High throughput; Mutagenesis; Mutant libraries; Pichia pastoris; Screening