2-1. Materials
2-1-1. Vector and Expression System
Recombinant AGHO and AGHOmutants genes were inert in plasmid pET30b without a S-tag (Appendix 3) in E. coli BL21(DE3).
2-1-2. Reagents
The chemicals were purchased from Merck and Sigma.
Spectra/Por molecularporous membrane tubing was obtained from Spectrum Medical Industries, Inc. HiTrap-chelating column was purchased from Amersham Biosciences. Bradford’s reagent was purchased from Bio-Rad. DNA Ladder and protein molecular weight marker were purchased from MBI. Prestained protein Ladder was purchased from Fermentas. Polyvinylidine diflutoide (PVDF) was obtained from Milllipore. All restriction enzymes were purchased from New England Biolabs, Inc. PufTurbo DNA polymerase was purchased from Merck. All other reagents and chemicals used in the experiments were reagent grade.
2-2. Methods
2-1-1. Construction of Plasmids
The wild type AGHO in pUC-T and the expression vector
pET30(-S)/AGHO was constructed previously in our laboratory [27, 34, 35]. The NotI fragment of wild type of AGHO gene (2069 bp) was purified from agarose gel and recovered using Gel/ PCR DNA
Fragments Extraction Kit (GENEAID). The purified DNA fragment was then used to replaced the NotI fragement of AGHO gene in
pET30(-S)/AGHO. The wild type AGHO in pGEM-T easy vector was constructed for the site-directed mutagenesis.
2-2-2. Site-Directed Mutagenesis
A156D, A156D/L158W, A156S/P157G, A156S/P157G/L158D, L158D mutants of AGHO were generated using QuickChangeTM Site-Directed Mutagenesis protocol (STRATAGENE) with pGEM-T easy/AGHO as a template. The reaction reagents contained 50 ng DNA templates, 125 ng forward and reverse primers, 2.5 U PufTurbo DNA polymerase, and 0.5 mM dNTP in a final volume of 50 µL. The PCR condition was set following the protocol of manufacturer. Then add 10 U Dpn I restriction enzyme into the resulting PCR product to remove the original methylated template. The reaction was performed by incubating at 37 ℃ overnight. The Dpn I-treated PCR product (15 µL) was then transformed into DH5α competent cells and screened for mutants. Furthermore, the mutations of AGHO were confirmed by DNA sequencing.
2-2-3. E. coli Expression and Purification of AGHO and AGHOmutants The recombinant wild type AGHO and its mutants were
overexpressd in E. coli BL21(DE3) cells carrying plasmid pET30b (-S).
A single colony was picked from a freshly streaked plate (LB-agar, supplemented with 25 µg/mL kanamycin), incubated for 12 hours at 37
℃, and then inoculated into 200 mL of LB medium. Flask cultures were grown at 37 ℃ and shacked at a speed of 150 rpm until the cell density reached A600nm =0.4~0.6. Cells were induced with 50 µM
isopropyl-1-thio-β-D-galactopyranoside (IPTG) and cultivated at 25 ℃ for 8 hours. Cells then were harvested by centrifugation at 6,000 rpm for 30 minutes.
The cell pellets were resuspended in 5 volumes of buffer A (50 mM potassium phosphate buffer, pH 6.8) and then disrupted on ice by
ultrasonic disintegration with the sonic dismembrator (550, Fisher Scientific). The resulting lysates were centrifuged at 14,000 rpm for 30 minutes to remove insoluble particulates. The supernatants were first fractionated with ammonium sulfate (1-50%). The precipitates of 50%
(w/w) ammonium sulfate were centrifuged at 10,000 rpm for 30 min and then dissolved in 1 mL buffer A. The resuscitations were dialyzed against buffer A at 4 ℃ for 12 hours and the buffer was renewed every 4 hours.
The protein solutions were then applied to a 1 mL pre-packed HiTrap-chelating column (Amersham Biosciences). The column was prepared following manufacturer’s protocol. Briefly, the column was washed with 5 mL distilled water prior to the recharging of Ni ions by loading 1 mL Charge buffer (100 mM NiSO4). Column was washed with 1 bed volume distilled water to remove the unbound metal ions. After
column preparation, the column was equilibrated with 5 bed volumes of binding buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM
imidazole). Samples were centrifuged at 14,000 rpm for 15 min prior to loading on the column. Column was then washed with 10 bed volumes of binding buffer. To further remove non-specifically bound proteins the column can be washed with binding buffer containing 60 mM imidazole.
The bound proteins were eluted with binding buffer containing 500 mM imidazole. Last but not least, the eluted protein was dialyzed overnight against buffer B (buffer A containing 50 µM CuSO4) to allow the
formation of metal-reconstituted proteins (including TPQ formation and Cu(II) binding). The unbound or weakly Cu(II) was removed from the protein by dialyzing with buffer A for 2~3 buffer changes (4 hours for each buffer change).
2-2-4. Protein Concentration Determination
Protein concentration was determined by Bradford protein assay (Bio-Rad) using bovine serum albumin as a standard. Briefly, the assay was performed in a constant reaction protocol by mixing 800 µL ddH2O, 200 µL Bradford reagent, and 2 µL eluted protein solution. The mixture was vigorous vortexed and incubated under room temperature for 10 minutes. Determine the absorbance of mixtureat wavelength 595 nm on the glass cuvette on a spectrophotometer (Hitachi U-3010). Calculate the protein concentration through intrapolation of a standard curve plotted on a same machine.
Amine oxidase activity was determined spectrophotometrically by monitoring H2O2 production through a coupling assay by horseradish peroxidase (HRP) using DMAB (3-dimethyl-aminobenzoic acid) and MBTH (3-methyl-2-benzothiazolinone hydrazone) as substrates [36]. A DMAB-MBTH conjugated purple indamine dye formed during reaction can be measured at absorbance of 595 nm. Assays were carried out in 2 or 20 µg enzyme reaction with amine substrate in the detection buffer (2.5 U horseradish peroxidase, 2 mM DMAB, and 0.04 mM MBTH in 50 mM sodium phosphate buffer, pH 7.4) in a total volume of 1 mL at 30 °C for 5 minutes. Absorbance measurement was obtained with Hitachi U-3010 using quartz cuvettes with 1 cm path length. Calculate the value of enzyme activity through the slope plotted of UV Solutions 2.1 on the same mechanism. Blank assay medium did not contain substrates.
2-2-6. H2O2 Standard Curve
The procedure is the same as that of activity assay except that amine oxidase and substrates were replaced with H2O2 in amounts from 0.1 to 15 nmoles. The standard curve of H2O2 concentration and O.D.
was illustrated in Figure 1.
2-2-7. Kinetic Measurement
Substrate stock solutions were freshly prepared in distilled water.
The concentration of substrate in the kinetic study varies based on type of substrate due to substrate inhibition effect. The oxidation of
substrates was determination by coupled assay (as described in Activity
Assay). The data were fitted non-linearly by at least six or more substrate concentrations. The curve fitting was performed using Michaelis-Menten equation 1 on the SIGMA plot program Enzyme Kinetics Module 1.1.
V=Vmax / (1+Km/[S]) (1) And the substrates demonstrating substrate inhibition were using equation 2:
V=Vmax / (1+Km/[S] +[S]/Ki) (2)
2-2-8. Electrophoresis and Redox-Cycling Staining
The protein solutions were separated on a 10% SDS
polyacrylamide electrophoresis gel (SDS-PAGE). All SDS-PAGE was performed with a Bio-Rad Mini Protein II apparatus. Enzyme samples were boiled for 5 min in the presence of 100 mM dithiothreitol before loading onto the SDS-PAGE. The electrophoresis was performed at 100 Volt for 20 minutes first, followed at 140 Volt for another 1.5 hours.
After electrophoresis, the gel was stained with 0.1% Coomassie Blue.
The redox-cycling approach provides a tool to reliably determine the quinone content of proteins for the study of the biological
significance of this process and the factors that affect protein quinolation. The redox-cycling staining with NBT-Glycinate was determined as previous described [37]: the proteins were separated on 10% SDS-polyacryamide gels and transferred to a polyvinylidine difluroide (PVDF) membrane on a Bio-Rad Mini Trans-Blot
Electrophoretic Transfer Cell soaked in an ice-cold transfer buffer (25
current of 200 mA for 2 hours. After electro-blotting, the membrane was immersed in the Glycinate/NBT solution (0.24 mM Nitroblue
Tetratzolium in 2 M potassium glycine, pH 10) for 30-45 minutes in the dark. The quinoproteins would be stained as blue-purple bands on the membrane.