title: Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example.
abstract: Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Li
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Liu
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.|Department of Urology, the First Affiliated Hospital of Chongqing Medical University, No.1, Youyi Road, Chongqing, 400016, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.affiliation: Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China. liaofeish@yeah.net.
authors.forename: Fei
authors.identifier: http://orcid.org/0000-0001-7671-6871
authors.initials: F
authors.lastname: Liao
mesh_terms: D000468:Alkalies / Q000737:chemistry; D001192:Arylsulfatases / Q000235:genetics / Q000302:isolation & purification*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity
chemical_list: D000468:Alkalies; D050505:Mutant Proteins; D001192:Arylsulfatases
keywords: Alkaline lysis; Directed evolution; High-throughput screening; Positive mutants; Pseudomonas aeruginosa arylsulfatase