N succinyl ala leu pro phe p nitroanilide

Recombinant His_6x-OsCYP19-4∆SP (OsCYP19-4 29–208 amino acids, corresponding to the mature protein) was prepared using the pET28a expression vector. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the 20kDa recombinant protein was purified using the ExiProgen^TM protein synthesizer (Bioneer, Daejeon, Korea). The PPIase activity of recombinant OsCYP19-4 mature protein was assayed using the tetrapeptide substrate Suc-AAPF-pNA (N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; Sigma-Aldrich, Ontario, Canada) (Fischer et al., 1984 (link)). All reagents were pre-equilibrated to 4 °C. In a 1mL cuvette, 50 or 100nM OsCYP19-4 recombinant protein was mixed with 100 μL α-chymotrypsin (60mg mL^–1 in 1mM HCl; Sigma-Aldrich, Ontario, Canada) and the volume was adjusted to 975 μL with an assay buffer (40mM HEPES-KOH, pH 8.0, 100mM NaCl, 2mM MgCl₂, 1mM EDTA). The reaction was initiated by the addition of 25 μL substrate (4mM tetrapeptide in 470mM anhydrous LiCl dissolved in trifluoroethanol). Changes in absorbance due to released p-nitroaniline were monitored over a 300 s period, at 390nm, at 10°C, in a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) with a thermostatically controlled cuvette holder. CsA, a PPIase inhibitor, was added at a concentration of 1 μM. The experiments were performed three times with different preparations of proteins.

The GhCYP-3 ORF was cloned into expression vector pET-32a (+) (Novagen, Darmstadt, Germany) with forward primer CYP-Bg-F and reverse primer CYP-Sa-R (Additional file 1: Table S1), which will introduce the BglII and SacI site into the 5′ and 3′ end of the ORF, respectively. To induce the expression of GhCYP-3 protein with a His-tag in E. coli BL21(DE3) (TransGen Biotech, Beijing, China), a final concentration of 1.0 mmol·L⁻¹ isopropyl-β-D-thiogalactopyranoside (IPTG) was added into the culture when the OD₆₀₀ value reached 0.4–0.6, and the culture was allowed to continue growing for 4 – 6 h before harvesting. The proteins were separated on a SDS–PAGE gel and detected by western blot using anti His-Tag mouse monoclonal antibody (1:5000; CW Biotech, Beijing, China). The recombinant protein was purified using a 6×His-Tagged Protein Purification Kit (CW Biotech, Beijing, China). The PPIase activity of the recombinant protein was assayed in vitro using the tetrapeptide substrate Suc-AAPF-pNA (N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; Sigma-Aldrich, Ontario, Canada) in a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) as described by Yoon (2016) [12 (link)]. Three biological and two technical replicates were analyzed for all measurement.