Strains, media, and expression plasmid
Escherichia coli TOP10F' (Invitrogen) was used for plasmid manipulations, and the S. cerevisiae strain MNN10 (MATa, mnn10, leu2Δ0, his3Δ1, met15Δ0, ura3Δ0) was used for protein expression. E. coli cells were grown in Luria-Bertani medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride) at 37°C. Yeast cultures were grown in YPD (1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal (SD) medium (0.67% yeast nitrogen base supplemented with the appropriate amino acids and 2% fructose) at 20 or 30°C. The expression vector used in this work was pYEX-S1 (pS1, Clontech), a yeast-E. coli shuttle vector containing the phosphoglycerate kinase (PGK) promoter, the E.coli Ampr gene, and the yeast selectable marker URA3.
Construction of the full-length RoGA construct
The full-length cDNA encoding RoGA was kindly provided by Simpson Biotech, and the set of primers F-RoGA and R-RoGA was used to clone the RoGA gene. Both primers included an additional EcoRI site at their 5'-ends. The sequences of all the primers used are listed in Table 4. PCR amplification was carried out with KlenTaq-1 DNA polymerase (Clontech). The 1.8-kb PCR product was gel-purified and subcloned into yT&A TA cloning vector (Yeastern Biotech) to generate a pT-RoGA plasmid, which was further digested with EcoRI and then subcloned into pS1 at the same enzyme site to generate a pS1-RoGA plasmid.
Construction of deletion mutants of RoGA
A series of deletion mutants was generated by a PCR-based technique using the pS1-RoGA plasmid as the template. For the C-terminal truncation clone GAΔ168–604, which lacks the catalytic domain, the template was amplified using the primer pair F-RoGA and R-GAΔ168–604. For GAΔ132–604, which lacks both the linker region and the catalytic domain, the template was amplified using primers F-RoGA and R-GAΔ132–604. The resulting PCR products were separated on a 1% agarose gel and subcloned into pS1 as described above. The internal deletion clone GAΔ132–167, which lacks the entire linker sequence, was generated by fusing an N-terminal fragment to a C-terminal fragment with a two-step PCR approach. In the first step, F-PS1 paired with R-GAΔ132–167 and F-GAΔ132–167 paired with R-PS1 were used to generate PCR products of 516 bp and 1517 bp, respectively. The PCR products thus obtained shared overlapping sequences that could anneal in a secondary PCR. In the second step, the two first-stage PCR products were purified, combined and reamplified together with the external primers F-PS1 and R-PS1. The generated PCR product was subcloned into pS1 and sequenced for confirmation. Similar technologies were employed to obtain the other internal deletion clones with the following primer pairs: GAΔ26–131, F-GAΔ26–131 and R-GAΔ26–131; GAΔ26–145, F-GAΔ26–145 and R-GAΔ26–145; GAΔ26–160, F-GAΔ26–160 and R-GAΔ26–160; GAΔ26–167, F-GAΔ26–167 and R-GAΔ26–167. All primer sequences are listed in Table 4.
Halo assay for GA activity
S. cerevisiae transformants were patched on YPD agar plate containing 0.5% (w/v) soluble starch. Secreted GA activity was detected by observing halo formation on agar plates containing iodine solution (0.01%).
Purification of secreted proteins from S. cerevisiae
Yeast strains were transformed using the one-step transformation method as described . The transformed yeast was cultivated in SD medium for 3 days at 30°C, and the supernatant of the culture medium was concentrated and dialyzed against 10 mM NaOAc (pH 4.5) using an Amicon stirred-cell concentrator (Millipore) equipped with a PM-10 membrane (10-kDa cut-off). For purification, the concentrated sample was loaded onto a 5-mL HiTrap SP cation-exchange column (Amersham Pharmacia Biotech) that was pre-equilibrated with the same buffer used for dialysis. The column was washed with 5 column volumes of 10 mM NaOAc buffer and then eluted with 10 volumes of a linear gradient of NaCl from 0 to 1 M in the same buffer at a flow rate of 2 mL/min. Fractions in 1-mL volumes were collected, and protein peaks were monitored by UV absorption at 280 nm. The fractions containing the desired proteins were pooled, dialyzed, and concentrated. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent kit (Pierce), with BSA as the reference standard.
Electrophoresis and Western blot analysis
SDS-PAGE was performed according to the method of Laemmli  using 10 or 15% (w/v) polyacrylamide gels. The protein bands in the gel were revealed by staining with Coomassie Brilliant Blue R-250. In Western blot analysis, extra- and intra-cellular samples were separated by SDS-PAGE and transferred electrophoretically onto PVDF membranes. The resulting Western blots were incubated with 3% BSA in TBS as the blocking solution and probed with polyclonal anti-RoGA antibody, used at a 1:5000 dilution. The secondary antibody was the horseradish peroxidase-conjugated anti-rabbit IgG, diluted to 1:5000. The bound complexes were detected with ECL reagents (ECL kit, Pierce) and exposure to X-ray film.
Mass spectrometric analysis
Molecular mass determination of the recombinant proteins was performed by Liquid Chromatography/Mass Spectrometer (LC/MS). The intact proteins (100 pmol) were acidified with 0.1% formic acid in 50% (v/v) acetonitrile, and the data were acquired over the 800–1800 m/z range under normal scan resolution. The original electrospray mass spectrum with multiply charged ion series was deconvoluted to give a mass spectrum.
N-terminal peptide sequence analysis
For N-terminal amino acid sequencing, purified protein was separated by SDS-PAGE, and electrophoretically transferred to a PVDF membrane. The blotted proteins were visualized using Coomassie Brilliant Blue R-250. After destaining, the blots were rinsed in deionized water and air-dried. N-terminal sequencing was performed by automated Edman degradation using an Applied Biosystems model 494 Procise sequencer at the National Taiwan University.
Peptide N-glycosidase F (PNGase F, New England Biolabs) and Jack bean α-mannosidase (Sigma-Aldrich) were used in enzymatic deglycosylation of the recombinant proteins according to the manufacturers' instructions. After enzymatic treatment, the samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue R-250 to detect any differences in protein migration.
Site-directed mutagenesis of amino acid residues in the linker
Site-directed mutagenesis was performed with a PCR-based technique to change Thr165 to Ala and Asn167 to Asp. The primers used were as follows: universal primers, F-PS1 and R-PS1; mutagenic primers F-T165A and R-T165A for the Thr165→Ala substitution; and F-N167D and R-N167D for the Asn167→Asp substitution. All primer sequences are listed in Table 4.
The adsorption of the SBD to granular corn-starch (Sigma-Aldrich) was performed as follows. The purified SBD-containing protein at a concentration ranging from 1.0 to 7.5 μM was mixed with 1 mg of prewashed corn-starch in a final volume of 1 mL in 10 mM NaOAc, pH 4.5. After incubation at 25°C for 16 h, the starch was removed by centrifugation at 16,000 g for 3 min at 4°C, and the amount of unbound protein remaining in the supernatant was determined by the BCA assay. The amount of adsorbed protein was calculated from the difference between the initial and unbound protein concentrations. The maximal amount of bound protein (Bmax) and the dissociation constant (Kd) were determined by fitting to the non-linear regression of the binding isotherms, and the following equation was used for saturation binding with one binding site: B = BmaxF/(Kd+F), where B (μmol) represents the bound protein, Bmax (μmol) is the maximal amount of bound protein, F (μmol) is the free protein in the system, and Kd (μmol) is the equilibrium dissociation constant. The units of calculated Bmax and Kd were converted to micromoles per gram and micromolar, respectively.
GA activity toward soluble starch
Substrate was prepared by boiling 1% soluble corn-starch in 10 mM NaOAc, pH 4.5. The amount of glucose liberated from the starch by enzymatic activity was determined using the glucose oxidase/peroxidase kit (Sigma-Aldrich). For each reaction, 50 μL of substrate solution was equilibrated in a 37°C water bath for 5 min, and the assay was initiated by adding 50 μL of appropriately diluted enzyme solution. After incubation at 37°C for 5 min, 200 μL of kit solution was added to each sample. The reaction mixtures were incubated for 5 min at 37°C and stopped by the addition of 200 μL of 6 M H2SO4. The absorbance was recorded at 540 nm using a U-3310 spectrophotometer (Hitachi). One unit of enzyme activity was defined as the amount of enzyme that produces 1 μmol glucose/min under the assay conditions described above.
Effect of temperature and pH on the activity of GA
Thermal stability of the enzyme was determined by pre-incubating the purified protein for 30 min at various temperatures ranging from 20 to 65°C. At time intervals, the residual activity was measured with the glucose assay kit as described above. Stability of the recombinant protein at different pH values was studied by incubating the enzyme in various buffers with pH values ranging from 2.0 to 11.0 for 2 h and then measuring the residual GA activity. The buffers used were 0.1 M glycine-HCl (pH 2.0, 3.0), 0.1 M sodium acetate (pH 4.0, 5.0), 0.1 M potassium phosphate (pH 6.0, 7.0), 0.1 M Tris-HCl (pH 8.0), and 0.1 M glycine-NaOH (pH 9.0, 10.0, 11.0).
Circular dichroism studies
Circular dichroism measurements were carried out with an AVIV model 202 spectropolarimeter (Aviv Associates, Lakewood, NJ). Far-UV wavelength scans were recorded from 200 to 260 nm with a bandwidth of 1.0 nm, using a 0.1-cm path length cuvette at 25°C. Each spectrum was an average of three consecutive scans and was corrected by subtracting the buffer spectrum. Thermal denaturation experiments were performed by increasing the temperature from 25 to 96°C, allowing temperature equilibration for 1.5 min before recording each spectrum. All of the data are expressed in terms of mean residue ellipticity, [θ]m.r.w., calculated by the equation: [θ]m.r.w. = (100θobs)/(nlc), where θobs is the observed ellipticity in degrees, n is the number of amino acids, l is the length of the light path in centimeters, and c is the molar concentration of the protein.