E coli bl21 de3

P. gulae genomic DNA was isolated as previously described [3 (link)] and used as a template for amplifying the entire fimA gene by PCR. The primers used for PCR were constructed from the sequences of the fimA genotypes [3 (link)] as follows: type A forward primers, 5′-GCG CGC GAA TTC GAG ATG AAA AAG ACT AAG-3′ and reverse primers, 5′-GCG CGG TTT AAG CTT TGA TTA CCA AGT AGC-3′; type B forward primers, 5′-GCG AAC GGA TCC AAG ATG AAA AAG ACT AAG-3′ and reverse primers, 5′-CGC TCT CTC GAG AGC TGA TTA CCA AAT-3; type C forward primers, 5′- ATC GAT ATC CAC TTT TAA AAC AAA AAA GAG-3′ and reverse primers 5′-TTT AGT CGT TTG ACG GGT CGA TTA CCA AGT-3′. Each amplified DNA was cloned into pGEM-T Easy Vector (Promega, Madison, WI), then digested with the appropriate restriction enzyme for ligation into the pET 42a (+) glutathione S-transferase (GST) gene fusion-protein expression vector (Novagen, Madison, WI, USA). The ligated vector was transformed into E. coli DH5α and colonies were selected on LB agar plates containing 50 µg of ampicillin/ml. Plasmid DNA was obtained using the Wizard® Plus SV Minipreps DNA Purification System (Promega). Each recombinant plasmid was transformed into E. coli BL21 (DE3) (Nippon Gene) and the colonies were selected as described above.

A pET23a-hTau40 gene was constructed by ligation of a synthesized hTau40 gene optimized for E. coli expression (Thermo Fisher, MA, USA) with a DNA fragment obtained from the expression vector pET-23a(+). Human Tau40 was expressed in E. coli BL21(DE3) (Nippon gene Co., Ltd, Tokyo, Japan) transformed with the pET23a-hTau40. Cells were disrupted by ultrasonication in purification buffer (50 mM Tris-HCl, pH 7.4, containing 2mM EDTA, 2mM DTT, and 0.2 mM PMSF) and centrifuged at 22,000 × g for 20 min. NaCl (final 200 mM) was added to the supernatant and heated at 85 °C for 10 min. After centrifugation (22,000 × g, 20 min), streptomycin sulfate (final 2.5%) was added to the supernatant and centrifuged at 22,000 × g for 20 min. Supernatants were dialyzed overnight and applied onto a CM sepharose column (GE Healthcare Life Sciences, Buckinghamshire, UK) with 50 mM Tris-HCl containing 2mM EDTA and 2mM DTT. Samples were eluted with a linear gradient of 0–0.5 M NaCl. The sample solution was dialyzed by 0.05 mM HCl and lyophilized. Molecular weight of hTau40 was assessed by SDS-PAGE and ESI-MS.