Comparative characterization, expression pattern and function analysis of the
12-oxo-phytodienoic acid reductase gene family in rice
Wenyan Li1, Feng Zhou2, Bing Liu1, Dongru Feng1, Yanming He1, Kangbiao Qi1,
Hongbin Wang1* and Jinfa Wang1
1,
State Key Laboratory for Biocontrol and Key Laboratory of Gene Engineering of Ministry of
Education, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, P. R. China
2,
*
College of Life Science, South China Agricultural University, 510642, Guangzhou, P. R. China
Corresponding author
Hongbin Wang, Tel/Fax: +862084039179; E-mail: wanghb@mail.sysu.edu.cn
1
Fig. S3 The expression of GST-OsOPR recombination proteins. A Coomassie Brilliant
Blue R250-stained SDS-polyacrylamide gel is shown. Protein standards are shown (lane
M), and their masses are indicated in kDa. Lanes 1-3, Lanes 4-6, Lanes 7-9, Lanes 10-12,
Lanes 13-15 and Lanes 16-8, show the crude cell extract, the cell pellet, the supernatant
of
GST-OsOPR08-1
(OsOPR7),
GST-OsOPR06-1
(OsOPR6),
GST-OsOPR04-1
(OsOPR11), GST-OsOPR02-1(OsOPR8) and GST-OsOPR01-1(OsOPR10), respectively.
Methods for isolation, purification and renaturation of inclusion bodies
MATERIALS
 Reagents and Solutions
10 × PBS: 1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4
(pH 7.3)
Buffer A: 1 × PBS + 0.1% Triton X-100 + 0.1 mM Dithiothreitol (prepare just before use)
Inclusion body washing buffer: Buffer A + 2% Deoxycholic acid
Inclusion body solubilization buffer: Buffer A + 0.3% N-Lauroylsarcosine
Inclusion body renaturation buffer: Buffer A
0.1 M dithiothreitiol (DTT): To 154 mg DTT (Sigma CAS D0632) dissolved in water,
constant volume to 10 ml, and store at -20 ℃ and dark condition.
5 M NaCl: To 146.1 g NaCl dissolved in water, constant volume to 500 ml.
20% (w/v) N-Lauroylsarcosine (SKL): To 10 g SKL (Sigma CAS L5125) dissolved in
2
water, constant volume to 50 ml.
20% (w/v) Deoxycholic acid (DOC): To 10 g DOC (Sigma CAS D6750) dissolved in
water, constant volume to 50 ml.
Dialysis tubing: (MMCO 8000~14000, D36mm)
Centrifuges: BECKMAN Avanti J-25 (4°C)
METHOD
 Isolation and purification of inclusion bodies
1. Collecting bacteria: Centrifuge 1 liter of the cell culture of E. coli expressing the fusion
protein at 7000 rpm for 15 minutes at 4°C in preweighed centrifuge bottles.
2. Bacterial ultrasonication: Remove the supernatant and add 5 ml buffer A per gram
(wet weight) of E. coli. Resuspend the pellet by gentle vortexing or by stirring with a
polished glass rod, and then break bacterial cell with the ultrasonic instrument (SONICS
VC130PB; parameters: 40 mV, ultrasonic 10s, intermittent 10s, continued to 15min).
3. Inclusion body recovery: Centrifuge of the cell disruption liquid at 4 ℃ for 15 min at
12000 rpm.
4. Inclusion body washing: Remove the supernatant and resuspend in 10 ml inclusion
body washing buffer at room temperature for at least 10 min.
5. Inclusion body purification: Centrifuge of the suspensions at 4 ℃ for 10 min at 12000
rpm. Remove the supernatant and repeat steps 4 and 5.
 Solubilization of inclusion bodies
6. Inclusion body solubilization: Add 10 ml inclusion body solubilization buffer and store
the solution for at least 1 hour at room temperature.
7. Inclusion body protein collection: Centrifuge of the soluble protein suspension at 4℃
for 10 min at 12000 rpm and take the supernatant.
 Renaturation and purification of proteins
8. Protein renaturation: The denatured and soluble protein was diluted 100 times into the
inclusion body renaturation buffer and filled into the dialysis tubing. After dialyzing as
fully stirring at 4℃ for 8 hours, replace the fresh buffer and repeat 2-3 times. To ensure
the correct folding of denatured GST-OsOPR fusion proteins, 1 μM cofactor FMN
(prepare just before use) was added into the renaturation buffer.
Pre-treatment of dialysis tubing:
1). Cutting the appropriate length of the dialysis tubing (10-20 cm).
2). Boiling the dialysis tubing in the large volume of 2% (W/V) sodium bicarbonate and 1 mmol/L
EDTA (pH8.0) for 10 minutes.
3). Washing the dialysis tubing with distilled water thoroughly.
4). Boiling the dialysis tubing in 1mmol/L EDTA (pH8.0) for 10 minutes.
5). Cooling and then storing the dialysis tubing at 4℃. (Note: The dialysis tubing must be
immersed in the solution and following steps would need to be wearing gloves when access to
dialysis tubing.)
6). Cleaning the Inside and outside of the dialysis tubing with distilled water before use.
9. Protein purification: After dialysis refolding, the fusion protein was purified according to
the Glutathione Sepharose™ 4B Kit (GE Healthcare, CAS 17-0756-01) instructions.
3