Pcold pros2 vector

The full-length coding sequence of MeSAUR1 was amplified with V2 primer pair (Table 1). The PCR product was ligated into the pCold Pros2 vector (TaKaRa, JPN) at the site of Nde I/Sal I, generating pCold Pros2-MeSAUR1. pCold Pros2-MeSAUR1 was introduced into Escherichia coli strain BL21 (DE3) for protein expression. E. coli cells containing pCold Pros2-MeSAUR1 were cultured in LB medium supplied with 100 mg/L Ampicillin at 37°C. When the OD₆₀₀ of the culture reaches 0.4–0.8, quickly cool the culture to 15°C in ice water, and then 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the cultures were incubated at 15°C, 120 rpm for 24 h. The cells were collected and resuspended in the BugBuster^® Protein Extraction Reagent (Novagen, GER) and incubated at 30°C for 1 h. The supernatant was collected and purified with Ni-Charged MagBeads (GenScript, United States). pCold Pros2 was expressed and purified as a control in accordance with the above methods. The purified protein was verified by SDS-PAGE and Western Blotting (Sambrook and Russell, 2001 ). Tag Anti-ProS2 (Takara, JPN) and Goat Anti-Mouse IgG/HRP (Boster, United States) were the primary and secondary antibodies used in Western Blot, respectively.

To get a large amount of MePHD1 protein, the full-length coding sequence of MePHD1 was amplified with P1 primer pairs and then fused to the Nde I/Sal I sites in the pCold Pros2 vector (TaKaRa, Tokyo, Japan), which generated the pCold Pros2-MePHD1. pCold Pros2-MePHD1 was introduced into Escherichia coli strain BL21 (DE3) competent cells for protein expression. The transformed strains were cultured in LB medium supplied with 100 mg/L Ampicillin, at 37 °C, and 250 rpm until the OD600 of the culture reached 0.4–0.8, and the bacterial cell fluid temperature cooled down to 15 °C. The cultures were incubated at 15 °C and 120 rpm, for 24 h, after adding 1.0 mM isopropyl β-d-1-thiogalactopyranoside. The soluble proteins of the cultured cells were extracted and purified by BugBuster^® Protein Extraction Reagent (Novagen, Darmstadt, Germany) and Ni-charged MagBeads (GenScript, Jiangsu, China), respectively. pCold Pros2 was expressed and purified as control. The purified protein was verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. When performing Western blotting, Tag Anti-ProS2 (TaKaRa, Tokyo, Japan) and Goat Anti-Mouse IgG/HRP (Boster, Wuhan, China) were used as primary and secondary antibodies, respectively.

The entire coding region of GmF3G2″Gt was amplified from cDNA of Harosoy and Nezumisaya by PCR using the KOD -Plus- DNA polymerase (Toyobo) with high PCR fidelity and primers containing enzyme sites for SacI and XhoI (Table 1). The PCR mixture contained 30 ng of genomic DNA, 10 pmol of each primer, 5 pmol of nucleotides, 2 mM of MgSO₄ and 0.5 unit of KOD –Plus- in 1 × KOD –Plus- Buffer supplied by the manufacturer in a total volume of 25 μl. PCR conditions were identical to those in a previous report [9 (link)]. The PCR amplicon was digested with SacI and XhoI and was cloned into the pCold ProS2 vector (Takara Bio). GmF3G2″Gt proteins were expressed and purified as described previously [22 (link)].