2.1. Enzymes and plasmids
Competent E. coli BL21 (DE3), the PCR cloning kit, PCR primers, NdeI and XhoI restriction enzymes and T4 polynucleotide kinase were obtained from Invitrogen. The pET15b vector was obtained from Novagen. Carbenicillin, ampicillin and chloramphenicol, isopropyl β-d-1-thiogalactopyranoside (IPTG), dithiothreitol (DTT) and tetracycline were obtained from Sigma. The LB medium and the bacterial growth-medium products were obtained from Qbiogene. HTP-Biogel resin was obtained from Biotech. The Hi-Prep 16/10 and Mono Q HR columns were obtained from Amersham Pharmacia Biotech.
2.2. Construction of the pET15b expression vector
The full open reading frame comprising the human 12R-LOX gene was PCR-amplified from the pQE80 plasmid using the forward primer CTCTCTCATATCGAA containing a 3′ NdeI site and the reverse primer TCTCTCTCTCTCGAG containing a 5′ XhoI site. The PCR-amplified product was digested with restriction enzymes (NdeI and XhoI) and the digestion product was purified and then added into similarly digested pET15b vector. The PCR-amplified 12R-LOX DNA was isolated from a 1.0% agarose gel using standard procedures. The clone thus obtained was confirmed by DNA sequencing.
2.3. Expression of LOX protein
LOXR/pET15b vector and chaperone plasmid PG-Tf2 were co-transformed into BL21 (DE3) cells. Transformed cells were selected for growth on LB agar plates supplemented with 50 µg ml−1 carbenicillin and 50 µg ml−1 chloramphenicol. An individual colony was selected to inoculate 200 ml LB medium containing 50 µg ml−1 carbenicillin and 50 µg ml−1 chloramphenicol. The culture was incubated at 310 K with constant shaking overnight. 200 ml overnight culture was then added to a litre of LB with 50 µg ml−1 carbenicillin and 50 µg ml−1 chloramphenicol and incubated at 310 K to an OD600 of 0.15. At this point 10 µg ml−1 tetracycline was added to the culture to express chaperone plasmid PG-Tf2 proteins. The culture was incubated at 288 K until an OD600 of 0.4 was reached. The culture was induced with 1 mM IPTG and incubated at 288 K with shaking for 24 h. After 24 h, the cells were harvested by centrifugation at 15 000 rev min−1 at 277 K for 10 min. The expression efficiency was verified by the SDS–PAGE technique (Fig. 1).
The expression efficiency was verified by the SDS–PAGE technique. Lane 1 contains SDS–PAGE markers (labeled in kDa). Lane 2 contains non-induced protein. Lanes 4–6 contain protein (LOXR with chaperone) induced with IPTG.
2.4. Purification of the 12R-LOX–chaperone complex
The cell pellet containing expressed 12R-LOX was resuspended in 100 ml lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 10 mM imidazole, 1% Triton X-100, 5 mM β-mercaptoethanol, 10% glycerol with two protease-inhibitor tablets). The resuspended cells were incubated on ice, sonicated for 10 min and centrifuged at 20 000 rev min−1 for 22 min. After centrifugation, the supernatant was loaded onto an immobilized Ni2+ metal-affinity chromatography (IMAC) column equilibrated with buffer A [50 mM Tris pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 1 mM imidazole] and eluted with an increasing concentration of buffer B [50 mM Tris pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 500 mM imidazole]. The purity of the protein was tested by SDS–PAGE. SDS–PAGE showed that bands containing the LOXR–chaperone complex eluted from the IMAC column at 50, 100, 250 and 500 mM imidazole. MALDI–TOF mass-spectrometric analysis indicated that the higher band running at approximately 58 kDa was the chaperone protein GroEL. These two proteins could not be separated, although a number of conventional chromatographic methods were attempted, including ion exchange, hydroxyapatite, hydrophobic interaction and gel filtration. High salt concentrations have been shown to disrupt protein–protein interactions. Fractions containing 12R-LOX and GroEL were pooled and concentrated to 3 ml. At this point, 2 M NaCl was added and the protein was incubated overnight at 277 K. After affinity-column purification, fractions containing 12R-LOX were pooled together and concentrated to 3 ml. 2 M NaCl was added and the protein was again incubated overnight at 277 K. The next day, the protein was loaded onto a gel-filtration column (Superdex 200) equilibrated with 50 mM Tris pH 8.0, 1 M NaCl and 5 mM β-mercaptoethanol. Fractions from the gel-filtration column were analyzed by SDS–PAGE (Fig. 2). Fractions from the gel-filtration column were pooled together and concentrated to 5 ml for thrombin digestion overnight at room temperature. Digested and undigested proteins were separated by IMAC for purification. Both digested and undigested proteins were concentrated to 1 ml for dialysis against 50 mM Tris pH 8.0, 50 mM NaCl, 5 mM β-mercaptoethanol at 277 K. The native gel (6% acrylamide) in Fig. 3 shows LOXR protein bound to chaperone protein. After concentration of the protein to 20 mg ml−1, ligand was added to the protein solution in a 1:1 molar ratio and it was set up for crystallization.
Figure 2 12% SDS–PAGE gel of the LOXR–chaperone complex obtained from a Superdex 200 gel-filtration column. Lane 1 contains molecular-mass markers (LMW, Pharmacia; labeled in kDa). Lanes 6–10 contain fractions containing LOXR with chaperone (more ...)
Figure 3 A native gel showing LOXR protein bound to chaperone. Lane 1 contains SDS–PAGE high-molecular-weight marker (labeled in kDa). Lane 2 contains digested LOXR protein bound to chaperone. Lane 4 contains undigested LOXR protein bound to chaperone. (more ...)
We obtained several crystals of the LOXR–chaperone complex with the ligand arachidonic acid as a substrate of the enzyme. Initially, crystallization conditions were screened by the sparse-matrix method (Jancarik & Kim, 1991
) using the Crystal Screen and Crystal Screen 2 kits (Hampton Research, USA). Typically, 2 µl protein solution at a concentration of 20 mg ml−1
was mixed with an equal volume of reservoir solution and equilibrated at 293 K against 1 ml reservoir solution. The complete process of crystallization was performed by the hanging-drop vapor-diffusion method (Ducruix & Giegé, 1992
) using Linbro multiwell tissue-culture plates. Crystallization trials showed needle-shaped crystals (Fig. 4). Subsequent optimization refined the crystallization conditions; the optimized composition of the precipitant was found to be 1.5 M
ammonium hydrogen phosphate in 0.1 M
Tris–HCl buffer pH 8.0. Under the optimized conditions, crystals appeared in 7 d and reached maximum dimensions that were suitable for X-ray diffraction.
Needle-shaped crystals of the LOXR–chaperone complex with ligand.
2.6. X-ray diffraction data collection and preliminary data analysis
The best crystal, with dimensions of 0.2 × 0.02 × 0.04 mm, was used for data collection. Prior to data collection, the crystal was soaked for several seconds in a solution containing 30% MPD as a cryoprotectant and was flashed-cooled to 100 K in a liquid-nitrogen cryostream. X-ray diffraction data collection was performed at 100 K using synchrotron radiation on beamline X9B at Brookhaven National Laboratory, USA. The crystal diffracted to 4 Å resolution and X-ray data were collected from a single crystal using a crystal-to-detector distance of 350 mm. X-ray diffraction data were indexed and processed using DENZO
and programs from the CCP
4 suite (Otwinowski & Minor, 1997
; Winn et al.
). Data-collection statistics are given in Table 1. The crystals belonged to the monoclinic system with space group P
and the unit-cell parameters were found to be a
= 138.97, b
= 266.11, c
= 152.26 Å, β = 101.07°. It has been found that the chaperone protein GroEL (57.16 kDa) remains as a heptamer (Hemmingsen et al.
) and two such heptamer rings encapsulate LOXR, giving a molecular weight for the complex of 880.58 kDa. With one complex molecule in the asymmetric unit, the Matthews coefficient (Matthews, 1968
) was calculated to be 3.1 Å3
and the solvent content was 60.5%. We were able to collect X-diffraction data to 4 Å resolution using synchrotron radiation and it is planned to solve the structure by the molecular-replacement method utilizing the E. coli
GroEL structure as a search model.