Plasmid mini prep kit

Sphingopyxis sp. USTB-05 used in this study was isolated and identified by us previously [45 (link)]. Trifluoroacetic acids and methanol were purchased from Dikma Technology Inc. (Lake Forest, CA, USA) and used for HPLC analysis. E. coli TOP10 and E. coli BL21 (DE3) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The recombinant and host strains were cultured in Luria–Bertani medium (tryptone 10 g, NaCl 10 g, yeast extract 5 g in 1 L H₂O) in a shaker at 200 revolutions per minute (rpm) at 30 °C. The vector pET30a (+), restriction enzymes Sac I and Not I, plasmid mini-prep kit, polymerase chain reaction (PCR) kit and Ni-NTA Sefinose (TM) Resin Kit were also obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Imidazole was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). NOD was purchased from Absin Bioscience Inc. (Shanghai, China) and stored at −20 °C. The purity of all other chemicals was no less than 99.7%.

E. coli JM109 and plasmid pPIC9K were used for gene cloning and propagation procedures. P. pastoris GS115 was used as a host for the expression of recombinant hPH20 and its mutants. PrimeSTAR Max DNA Polymerase, T4 DNA ligase, T4 polynucleotide kinase, restriction endonucleases, and protein markers were purchased from TaKaRa (Dalian, China). Ampicillin, G418 sulfate, and plasmid Mini Prep Kit were purchased from Sangon Biotech (Shanghai, China). The gel extraction kit and 10% Bis-Tris protein gel were purchased from Thermo Scientific (Shanghai, China). Hyaluronidase from bovine testicular tissue (BTH) was obtained from Sigma-Aldrich (St Louis, MO, United States). Other chemicals were obtained commercially and were of reagent grade.

Recombinant pGEX-4T-1/USTB-05-A/BL21(DE3) and pET30a(+)/USTB-05-C/BL21(DE3) were previously studied on MCs and used in this study for NOD biodegradation [30 (link),49 (link)]. E. coli TOP10 and E. coli BL21(DE3) were purchased from Sangon Biotech (Shanghai, China). The recombinant and host strains were cultured in Luria–Bertani (LB) medium [41 (link)] on a shaker at 200 rpm at an appropriate temperature. The vectors pGEX-4T-1 and pET30a(+), restriction enzymes BamHI, XhoI, SacI and NotI, the plasmid mini-prep kit and the polymerase chain reaction (PCR) kit were obtained from Sangon Biotech (Shanghai, China). Standard NOD was purchased from Enzo Science Inc., Farmingdale, NY, USA. All other chemicals were analytical grade.