Mutan super express km

Plasmid purification kits (QIAGEN Plasmid Maxi Kit, QIAprep Spin Miniprep Kit) were purchased from QIAGEN (Venlo, The Netherlands). Restriction enzyme, T4 polynucleotide kinase, alkaline phosphatase (E. coli C75), DNA ligation kit (DNA Ligation Kit Ver.1), DNA polymerase (TAKARA Premix Taq, EX Taq version), and Site-Directed Mutagenesis kit (Mutan^®- Super Express Km) were purchased from Takara Bio (Kusatsu, Japan). Heparin was purchased from Mochida Pharmaceuticals (Shinjuku City, Tokyo) and Block Ace from Sumitomo Dainippon Pharma (Osaka, Japan). Blue Sepharose 6-Fast Flow, 5 mL HiTrap Phenyl HP, and 5 mL HiTrap Q XL were purchased from GE Healthcare Japan (Tokyo, Japan). HE staining reagents were purchased from Muto Chemical (Tokyo, Japan). Mouse anti-α-SMA antibody (cat#:ab5694) was purchased from abcam (Cambridge, UK). All other reagents and solvents were commercially available special grade products, and the water used as the solvent was ion-exchanged water or Milli-Q water.

The plasmid pMAQΔc2, which was designed to express wild-type AQN as a fusion protein with maltose binding protein (MBP), was constructed based on pAQNΔC105 and pMAL plasmids (New England Biolabs, Ipswich, MA, USA) as described previously (Sakaguchi et al. [2008a (link)]).
To construct plasmids with a mutated aqualysin I gene, site-directed mutagenesis was performed following the ODA-PCR method (Mutan®-Super express Km; TAKARA BIO) using pMAQΔc2 as a template. The oligonucleotide primers (Sigma-Aldrich Life Science, Hokkaido, Japan) used for site-directed mutagenesis are shown in Table 2. The fragments containing either of the mutations listed in the table were inserted into the expression vector, pMAQΔc2. The names of the mutant plasmids are provided in the third column of Table 2 (D17N, etc.). The nucleotide sequences around the mutation sites as well as other parts of the gene were confirmed by DNA sequencing using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).