Bacterial strains and growth media
Caulobacter crescentus CB15 (ATCC No. 19089) was obtained from the American Type Culture Collection (ATCC). The E. coli strains used as the expression hosts and the gene engineering hosts were BL21 (DE3), DH5α, HB101, JM109, MV1184, Origami (DE3) and Tuner (DE3).
LB medium (1% Bacto Tryptone, 0.5% Bacto Yeast extract, 1% NaCl, pH 7.0) was used for culturing E. coli in the genetic engineering and in the preculture for protein expression. The solid medium contained 1.5% agar. Ampicillin (50 μg/ml) was added to the medium as needed.
Gene cloning and chemical reagents
Genetic engineering experiments were performed according to the procedure described by Sambrook and Russell (2001). The enzymes used for genetic engineering were purchased from TAKARA BIO INC. (Shiga, Japan) and used according to the manufacturer’s instructions. Bacto Tryptone, Bacto Yeast extract and peptone were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Other reagents and oligosaccharides used as substrates were of the highest quality available from Wako Pure Chemicals (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified.
Cloning of the GA gene from C. crescentus CB15 and construction of the expression plasmids
C. crescentus CB15 was grown in Caulobacter medium (peptone, 2.0 g; yeast extract, 1.0 g; MgSO4•7H2O, 0.2 g in 1 liter) at 30°C for 2–3 days, and the genomic DNA was extracted using ISOPLANT II (Nippon Gene, Tokyo, Japan).
PCR was performed as follows: 1 cycle at 94°C for 2 min followed by 30 cycles of the sequence at 94°C for 15 s, 63°C for 30 s and 68°C for 2.5 min were performed, using 25 ng of the genomic DNA as a template, with the forward primer 5′-CGCGGATCC GCGATGCGCACGTTGAAAAC-3′, and the reverse primer 5′-GGAATTC CTAGCGCGCGTACCGCGC-3′ (the BamHI and EcoRI restriction sites are underlined), and the PCR product was cloned into the BamHI and EcoRI sites of pUC119 vector to give pUC119-CauloGA. Next, PCR was performed using the pUC119-CauloGA as a template, with the forward primer 2, 5′-GCGAATTC GGCGCCTACGACCTGGGCCTATTCG-3′, and the reverse primer 2, 5′-CGGGATCC CTAGCGCGCGTACCGCGCCTTTACGGG-3′ (the EcoRI and BamHI sites, respectively, are underlined), to remove a putative signal peptide sequence that was predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/). The amplified PCR product was cloned into the EcoRI and BamHI site of the pEZZ18 vector (GE Healthcare, Tokyo, Japan) to yield the expression vector, pEZZ18-CauloGA. Expression vectors, pCold I-CauloGA and pCold TF-CauloGA, were constructed similarly using the pCold vectors (TAKARA) and pUC119-CauloGA plasmid. The nucleotide sequences were confirmed with the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
The nucleotide sequence of CauloGA is available in the DDBJ/EMBL/GenBank database under the accession number AB813000.
Expression, purification and activity measurement of CauloGA
The E. coli that was transformed with the pEZZ18-CauloGA expression plasmid was precultured overnight in LB medium at 30°C, and the preculture was inoculated in fresh 2 × YT-medium (1.6% Bacto Tryptone, 1% Bacto Yeast extract, 0.5% NaCl, pH 7.0) at 30°C for 24 h, harvested by centrifugation, suspended in 20 mM Tris-HCl (pH 7.0) containing 0.5 M NaCl and sonicated on ice using a Ultrasonic disruptor UD-201 (TOMY SEIKO, Tokyo, Japan). The supernatant obtained from the crude extract by centrifugation for 20 min at 20,000 × g was adsorbed onto an IgG Sepharose column (GE Healthcare, Tokyo, Japan) that was equilibrated with TS buffer [50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl]. The bound fusion proteins were eluted with TS buffer containing 40% (v/v) 1,4-dioxan (Kumari and Gupta 2012). The purity of CauloGA was confirmed by 9% SDS-PAGE (Laemmli 1970). The fraction containing the active enzyme was dialyzed against 20 mM Tris-HCl, 0.5 M NaCl (pH 7.0), and the purified protein was stored at 4°C.
The enzyme activity was measured at 40°C in 50 mM acetate buffer (pH 5.0) with 0.9 mM maltotriose as a substrate unless otherwise specified. The enzyme solution (50 μl) was added to 450 μl of the substrate solution, and the reaction mixture was incubated for an appropriate period. To halt the reaction, 50 μl aliquots of the mixture were removed from the reaction mixture and mixed with 450 μl of the stop solution, 1 M Tris-HCl (pH 7.0), at appropriate intervals, and the initial rate was estimated from the initial slope of the reaction time course. The amount of glucose liberated from the substrate was determined with the F-kit D-glucose (Roche Diagnostics Gmbh, Mannheim, Germany) using the glucose standard curve as a reference. One unit of GA was defined as the amount of the enzyme that liberates 1 μmol of glucose from the substrate in 1 minute. The protein concentration was measured using the micro-assay method (Bio-Rad Laboratories, Hercules, CA, USA), which is based on the Bradford method (Bradford 1976), using bovine serum albumin as a standard.
Polyclonal antibody against the CauloGA gene product expressed in E. coli as an inclusion body
The (His)6-tagged CauloGA was expressed in E. coli as an inclusion body using the pCold I vector system (TAKARA). The inclusion bodies were dissolved in 20 mM Tris-HCl containing 6 M guanidine hydrochloride, and the denatured CauloGA protein was purified on HisTrap™HP (GE Healthcare) and the purified denatured CauloGA was used as the antigen to raise the rabbit anti- CauloGA antibody that was prepared by TAKARA.
Western blotting analysis of CauloGA
The CauloGA expressed in E. coli using the pEZZ18-CauloGA expression plasmid was detected according to the procedure described in a previous report (Takeshima-Futagami et al. 2012). The CauloGA protein was detected using anti-CauloGA antibody and the HRP-conjugated goat anti-rabbit IgG (H + L) antibody (Invitrogen, Carlsbad, CA, USA) as the primary and the secondary antibodies, respectively. The bound antibodies were detected using the SuperSignal West Dura Trial Kit (Thermo Scientific, Rockford, IL, USA).
Determination of the anomer type of the products of the CauloGA reaction
Maltotriose as a substrate was dissolved in D2O containing 50 mM 2-morpholinoethanesulfonic acid (MES)-NaOH (pH 6.0). The enzyme was mixed with 4.5 mM maltotriose and incubated at 30°C. At the appropriate reaction time, the 1H-NMR spectra were measured using a JNM-ECX-400 (JEOL, Tokyo, Japan) spectrometer. The chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) (Cambridge Isotope Laboratories, Inc., Andover, MA, USA), as the internal standard.
Measurement of temperature- and pH-dependence of the activity, heat stability and pH stability of CauloGA
The temperature-dependence of the CauloGA activity was examined by measuring the initial rate as described above, at 15, 20, 25, 30, 35, 40 and 45°C. To determine the heat stability of CauloGA, the enzyme solution was incubated at various temperatures (4-60°C) for 60 min and cooled quickly, and the remaining activity was measured. The pH-dependence of the CauloGA activity was determined by measuring the initial rate at different pH values, ranging from pH 3.5 to 11, using 0.9 mM maltotriose as a substrate in various buffers as follows: 50 mM acetate buffer (pH 3.5-6.0), 50 mM MES-NaOH buffer (pH 5.0-7.0), 50 mM Tris-HCl buffer (pH 7.0-9.0) or 50 mM carbonate-NaOH buffer (pH 10-11). To study the pH-stability of CauloGA, the enzyme solution was placed in various buffers (pH 3.5-11) at 4°C for 60 min, and the remaining activity was measured.
Differential scanning fluorimetry (DSF) analysis
The experiment was performed using a Real Time PCR System (Mx3005p, Agilent Technologies, Santa Clara, CA, USA) and MxPro Software (Agilent Technologies). Tubes containing a mixture of 39 μl of a protein solution and 1 μl of Sypro Orange (Invitrogen) diluted to 100-fold with dimethyl sulfoxide were set in the PCR instrument and were subjected to the temperature scan at 1°C min-1 from 25°C to 95°C. The filter configurations were customized to accommodate the optimal excitation and emission wavelengths for Sypro Orange (Ex: 492/Em: 610 nm). The inflection points of the transition curve, indicating the melting temperatures (Tm) of the protein, were estimated from the sigmoidal curves of the fluorescence intensity using the Boltzmann equation and a graphics software package (DeltaGraph ver. 6, Nihon Poladigital K.K., Tokyo, Japan), according to the method of Niesen et al. (2007).