Activity Assays for Bacteriophage Endolysin PlyPy
噬菌体细胞内溶素PlyPy的活性分析
发布: 2014年09月20日第4卷第18期 DOI: 10.21769/BioProtoc.1233 浏览次数: 10668
评审: Kanika GeraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
Bacterial viruses (bacteriophages) escape and kill their host by degrading the bacterial peptidoglycan layer through the mechanism of enzymes called endolysins: peptidoglycan degrading enzymes. The method included here is useful for the initial characterization of any endolysin, regardless of the specific catalytic domain (as long as the activity results in a reduction in the optical density), in order to determine its optimal enzymatic (lytic) activity. This protocol is specific for the Streptococcus pyogenes phage endolysin PlyPy, but can be adapted for any peptidoglycan degrading enzyme.
Keywords: Phage lysins (噬菌体裂解酶)
Lysis (裂解)
Lytic activity (溶解活性)
Cell wall hydrolase (细胞壁水解酶)
Peptidoglycan cleavage (肽聚糖裂解)
Materials and Reagents
- Streptococcus pyogenes (S. pyogenes) strain MGAS 3 15
- Purified phage lysin PlyPy (or other lysins of interest)
- Brain heart infusion (BHI) broth (BD, catalog number: 237500 )
- Agar (BD, catalog number: 214010 )
- Tris (Thermo Fisher Scientific, catalog number: BP152-1 )
- HCl (Thermo Fisher Scientific, catalog number: A144S-500 )
- CaCl2.2H2O (Thermo Fisher Scientific, catalog number: C70-500 )
- MgCl2.7H2O (AMRESCO, catalog number: 0288-100G )
- ZnCl2 (Fluka, catalog number: 96468-50G )
- DTT (Sigma-Aldrich, catalog number: D0632-10G )
- Sodium acetate (Thermo Fisher Scientific, catalog number: S210-500 )
- NaCl (Thermo Fisher Scientific, catalog number: S271-3 )
- PlyPy activity buffer (see Recipes)
Equipment
- 37 °C incubator
- Spectrophotometer (Bio-Rad Laboratories, SmartSpecTM, model: 3000 )
- Table centrifuge (Eppendorf, model: 5810 R )
- pH meter (Thermo Fisher Scientific, AccumetTM, model: 15)
- 15 ml tubes (Falcon®, catalog number: 352097 )
- 50 ml tubes (Falcon®, catalog number: 352098 )
- Cuvettes (Sarstedt AG, catalog number: 67.742 )
- 96-well flat bottom plates (Falcon®, catalog number: 35-1172 )
- 96-well plate reader (Molecular Devices, SpectraMax Plus Reader)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lood, R. and Fischetti, V. A. (2014). Activity Assays for Bacteriophage Endolysin PlyPy. Bio-protocol 4(18): e1233. DOI: 10.21769/BioProtoc.1233.
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分类
微生物学 > 微生物-宿主相互作用 > 病毒
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 活性
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