A Practical System for High-Throughput Screening of Mutants of Bacillus fastidiosus Uricase.
For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl2, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate Vm/Kmby kinetic analysis of reaction curve for comparison. Vm/Kmwas resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Tao
T
Feng
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Xiaolan
X
Yang
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Deqiang
D
Wang
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Xiaolei
X
Hu
Central Laboratory, Yongchuan Hospital, Chongqing Medical University, No.439, Xuanhua Road, Yongchuan, Chongqing, 402160, China.
Juan
J
Liao
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Jun
J
Pu
Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
Xinyun
X
Zhao
Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA.
Chang-Guo
CG
Zhan
Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China. liaofeish@yeah.net.
Fei
http://orcid.org/0000-0001-7671-6871
F
Liao
D001407:Bacillus / Q000201:enzymology*; D004252:DNA Mutational Analysis / Q000379:methods*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D014503:Urate Oxidase / Q000737:chemistry / Q000145:classification / Q000235:genetics*
D014503:Urate Oxidase
Bacillus fastidiosus uricase; Cell lysate; Directed evolution; Error-prone PCR; High-throughput screening