Restriction endonucleases nde 1

Geobacillus stearothermophilus was purchased from Microbial Culture Collection Center of Guangdong Institute of Microbiology, China. Competent E. coli cells DH5α and BL21 (DE3) were purchased from TIANGEN Biotech (Beijing, China). Plasmid pET21a was preserved by our own laboratory. Restriction endonucleases Nde I and Xho I were purchased from ThermoFisher Scientific Co., Ltd. (Shanghai, China). Reagents related to Bst DNA pol LF gene cloning and site-directed mutagenesis were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The HisTrap^FF column from GE Healthcare (Piscataway, NJ, USA) was used for proteins purification. The chemicals used for HPLC were obtained from Sigma-Aldrich Chemical (St. Louis, USA). All chemicals used in this study were of analytical grade.

Competent E. coli cells DH5α and BL21(DE3) were purchased from TIANGEN Biotech (Beijing, China). Plasmids pTWIN1, pET28a-SUMO, and pET21a were preserved by our own laboratory. Restriction endonucleases Nde I and Xho I were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Prime STAR HS (Premix) LA Taq, DNA ladder Marker, and kits for DNA manipulation were purchased from TAKARA Biotechnology Co., Ltd. (Dalian, China). Protein ladder marker was obtained from Thermo Fisher Scientific (CA, USA). Pfu DNA polymerase was purchased from Promega (Madison, USA). hEGF coding region was synthesized by Huada Genomics Institute Co., Ltd. (Shenzhen, China). Commercial recombinant hEGF was purchased from Peprotech Co., Ltd. (Rocky Hill, USA). Mouse fibroblast Balb/c 3T3 cells were kindly given by Professor Yadong Huang from Jinan University. All chemicals used in this study were of analytical grade.

M. xanthus DK1622 (DSM 435) was obtained from the German Collection of Microorganisms and Cell Culture (DSMZ). Escherichia coli Mach1 cells (Life Technologies, Shanghai, China) were used for plasmid amplification and manipulation. E. coli strain BL21 (Invitrogen, Shanghai, China) was used as the expression enzyme. The pET30a (+) plasmid was used as vector for the heterologous overexpression. Primestar DNA polymerases were purchased from Takara (Dalian, China). Restriction endonucleases (NdeI, XhoI, and DpnI) and T4 ligases were purchased from Thermo Fisher Scientific (Shanghai, China). The Plasmid Extraction Kit and the DNA Gel Purification Kit were from Axygen (Beijing, China). DNA primers were synthesized by GenScript (Nanjing, China). Thin-layer chromatography (TLC) was performed using Merch 60 F254 precoated silica gel aluminium plates. Substrates and products were visualized by exposure to a solution of diphenylamine (0.1 M), phosphoric acid (10% v/v) and aniline (2% v/v), in acetone (DPA). Kanamycin and isopropyl-β-D-thiogalacto-pyranoside (IPTG) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other reagents used in this study were sourced from local chemical suppliers.