2.1 Expression of the V. alginolyticus pepD gene in E. coli
We used pET-28a(+) as the expression vector. Constructed pET-28a(+)-pepD plasmids were transformed into E. coli BL21(DE3)pLysS competent cells by means of the heatshock method, and the cells spread on an LBkan agarose plate and incubated at 37ºC for 12 to 16 hrs.
Colonies harboring pET-28a(+)-pepD were extracted and cultured in 3 ml LBkan medium for several hours, and then transferred into a 300 mL LBkan medium. 150 μL 1 M IPTG (final concentration was 0.5 mM) was added when the OD600 approached 0.5 ~ 0.6, and then the colonies incubated at 37ºC for another 6 hrs to induce the expression of PepD protein.
pET-28a(+) plasmids also were transformed into E. coli BL21(DE3)pLysS competent cells, following the same experimental procedure as for controls.
2.2 Purification of the expressed Vibrio alginolyticus PepD
After 6 hrs incubation at 37ºC, with rotary shaking, cells were collected by centrifugation at 6,500rpm for 30 min at 4ºC. The bacterial pallet was resuspended with 20 mL 20 mM Tris-HCl, 0.5 M NaCl, pH 6.8 buffer (buffer A). The resuspended cells were disrupted by sonication using a sonicator pulsing in a 2 sec on, 1 sec off pulsation cycle for the total sonication time of 3 min at 30% energy. The entire experimental sonication procedure was conducted on ice. The sonication steps were repeated at least 2 additional times. After sonication, the cell lysate was centrifuged at 9,500rpm for 30 min at 4ºC to remove the cell debris and intact cells. The supernatant was collected for further purification.
The supernatant was purified by affinity chromatography using a Ni-NTA column. One mL Ni-NTA resin was packed in a 20 mL plastic column and pre-equilibrated with 10 mL buffer A containing 20 mM imidazole (10 bed volume). The supernatant was loaded into the column and then washed with 10 mL buffer A containing 20 mM imidazole (5 bed volumes).
Five bed volumes of buffer A, consisting of 80 mM, 150 mM, 300 mM, or 500 mM imidazole, were used sequentially to elute the expressed PepD protein. Finally, the Ni-NTA column was washed with buffer A, containing 1 M imidazole. The eluted fractions were collected for SDS-PAGE analysis and enzymatic activity assay. By SDS-PAGE analysis, the high-purity eluted fractions were collected and dialyzed with 2 L 50 mM Tris-HCl pH 6.8 buffer for 2 hrs, followed by 3 L for 8 hrs. After enzymatic activity analysis, the purified proteins were stored at -80ºC to be ready for subsequent experiments. Note that PepD can be stored at -80 ºC for six months with no loss of of activity.
2.3 Protein concentration determination
The protein concentrations of purified proteins were measured using BCA Protein Assay Reagents. To each well of the F96 MicroWellTM plate was added a 20 μL sample mixed with 200 μL BCATM Working Reagents (BCATM Reagent A:BCATM Reagent B = 50:1). The reactions were incubated at 37ºC for 30 min in the dark. The absorbances of samples were measured at 562 nm on a Multiskan Ascent Microplate Reader. 2 mg/mL bovine serum albumin (BSA) stock and successive dilutions (1.5, 1.0, 0.75, 0.5, 0.25, 0.125, 0.025 mg/mL) served as standards, following the same procedure described above.
2.4 SDS-PAGE and Native-PAGE analysis
After expression and purification, gel electrophoresis was used to assess for protein expression level, purity, and molecular weight. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) on 12.5% gels (Table 2).
Each 10 μL sample was mixed with 2 μL 5X SDS-PAGE sample buffer and incubated at 95ºC for 5 min to denature proteins. Electrophoresis was performed with 1X SDS-PAGE running buffer at 90 Volts for 30 min, followed by 120 Volts for 1.5 hrs. The SDS-PAGE gel was stained with a stain buffer containing Coomassie Brilliant blue R-250 for 30 min and destained with destain buffer I (methanol/acetic acid/water = 4:1:5, v/v/v) for 20 min, followed by destain buffer II (methanol/acetic acid/water = 1.2:0.05:8.75) overnight.
Native-PAGE was performed to examine the native form of PepD. The purified and dialyzed protein fractions were separated by Native-PAGE on 7.5% gels (Table. 1). The experimental steps were similar to SDS-PAGE analysis, except that the gel contained no SDS and there was no denaturing treatment. Each 10 μL sample was mixed with 2 μL 5X Native-PAGE sample buffer, and immediately followed by an iced 1X Native-PAGE running buffer at 90 Volts for 3 hrs in a 4ºC circulating water bath. The proteins were stained and destained in the same way as for SDS-PAGE analysis.
Table 1: Solutions and volumes for the preparation of the SDS-PAGE and Native-PAGE separating gel and stacking gel
Separating gel Stacking gel
TEMED (mL) 0.028 0.014 0.014 0.014 0.014 0.014 6.8
10 % Ammonium persulfate
(APS)b (mL) 0.35 0.35 0.35 0.35 0.35 0.35 10
Total (mL) 35 35 35 35 35 35 10
a Replaced SDS with ddH2O while preparing Native-PAGE
bRecommended that it be prepared fresh and mixed at the end
2.5 Enzymatic activity assay of PepD
PepD activity was determined according to Teufel et al.[33] via the measurement of histidine, using an o-phthalaldehyde [16] reagent. The substrate, L-carnosine (β-Ala-L-His), was hydrolyzed to β-Alanine and L-Histidine. The fluorescence of the derivative of histidine with OPA was detected at λEx: 355 nm and λEm: 460 nm ( Fig. 6 ).
HN N
Fig. 6: Formation of a Schiff base by L-histidine and o-phthalaldehyde
20 μL purified enzyme (0.5 mg/mL) and 80 μL 50 mM Tris-HCl pH 6.8 buffer reacted with 0.5 mM L-carnosine over 20 min. Liberated histidine was derivatived by adding 100 μL OPA reagent, and incubated at 37ºC in darkness for 5 min. The reactions containing just buffer with L-histidine or L-carnosine reacting with OPA served as positive and negative controls, respectively. All reactions were carried out in triplicate. The fluorescence of the histidine derivatived with OPA was measured by Fluoroskan Ascent FL. (λEx: 355 nm and λEm: 460 nm).
2.6 Mutagenesis analysis of V. alginolyticus pepD
Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Appendix 2) to create the mutants. Mutagenic primers were designed and pET-28a(+)-pepD plasmids (wild-type) were used as the template: the PCR reaction was carried out via the nonstrand-displacing action of pfuTurbo DNA polymerase to extend and incorporate the mutagenic primers (Appendix 3), resulting in the nicked circular strands. The PCR mutagenesis reaction was performed in a 96-well GeneAmp® PCR System 9700 Thermal Cycler, as recommended by the manufacturer of PfuUltraTM High-Fidelity DNA polymerase. For each reaction, 100 ng of wild-type plasmid, 5 μL 10X Pfu polymerase buffer, 4 μL 2.5 mM dNTP mix, 1 μL of each 12.5 μM primer, 1 μL (2.5 U) Pfu polymerase and ddH2O were added to the final volume of 50 μL (Table 3). The PCR products with wild-type
and mutant plasmids were incubated with DpnI for 4 hrs at 37ºC to selectively digest the methylated, non-mutated parental wild-type plasmids. After DpnⅠdigestion, the mutant plasmid was transformed into E. coli XL1-Blue competent cells, with selection for kanamycin resistance. After successful mutagenesis, which was confirmed by restriction enzymes and DNA sequencing of plasmid, the desired mutant plasmids were transformed into E. coli BL21( DE3 ) pLysS competent cells for expression of the mutant pepD proteins.
Table 2: Reaction conditions and cycling parameters for the PCR mutagenesis reaction.
2.7 The His-tag-cleaved PepD
After purification of expressed Vibrio alginolyticus wild-type PepD (see 2.2), containing a His-tag at the N-terminal, the eluted PepD-containing protein fractions were incubated with thrombin (Sigma) in a cleavage buffer (40 mM Tris (pH 8.0), 300 mM NaCl, 2 mM CaCl2, and 5% glycerol) for 16 hours to digest the His-tag. After being subjected to benzamidine sepharoseTM6B (Pharmacia) to remove thrombin, the flow-through fractions were applied to another Ni-NTA column to collect and remove the His-tag fragments. The pooled native-form PepD proteins then were dialyzed with a 20mM HEPES buffer (pH 7.0), and stored at -80°C.
2.8 Metal ion effect of PepD activity
The His-tag-cleaved PepD protein first was dialyzed overnight with buffer containing 20 mM MES pH6.0 and 5 mM EDTA to remove divalent zinc ion (apo-Pepd). The apo-PepD was dialyzed twice with 20 mM MES pH6.0 and exchanged with 20 mM HEPES pH7.0 before adding various divalent metal ions. The apo-PepD protein concentration was adjusted to 0.01 mM. Metal ions - including MgCl2, MnCl2, FeCl2, CoCl2, NiCl2, CuCl2, and CdCl2 - were added into the final 20 mM HEPES buffer for metal exchange dialysis.
2.9 Enzyme kinetics
For determination of Vmax, Km, and kcat of the V. alginolyticus PepD, and to compare hydrolysis efficiency with the wild-type and mutant PepD, the method described by Csámpai et al.[47] was modified using High Performance Liquid Chromatography (HPLC) with a Fluorescence Detector (FLD). A system consisting of an Agilent 1100 Series Quaternary pump, Autosampler, Fluorescence Detector and Inertsil ODS-3 (7 μm, 7.6 mm×250 mm) column was used. The eluent system consisted of two components: eluent A was 0.05 M sodium acetate at pH 7.2, while eluent B was prepared from 0.1 M sodium acetate–acetonitrile–methanol (46:44:10, v/v/v) (titrated with glacial acetic acid or 1 M sodium hydroxide to pH 7.2). The gradient program was as described in Table 3. The fluent flow-rate was 0.8 mL/min at 30 ºC.
Table 3: The fluent gradient program
Step Time (min) A (%) B (%) 1 0 100 0 2 5 50 50 3 15 25 75 4 20 0 100
Different concentrations of L-carnosine (2, 1, 0.5, 0.25, 0.1 and 0.025 mM) were added as substrates to initiate enzymatic reactions. After 20 min incubation at 37 ºC, the samples were mixed with OPA reagent for 5 min incubation at 37ºC, then injected by autosampler.
Fluorescence of the histidine with derivatived OPA was measured by FLD (λExc: 355 nm and λEm: 460 nm). Various concentrations of L-histidine solution (0.1, 0.05, 0.025, 0.01, 0.005, and 0.0025 mM) derivatived with OPA reagent were detected, using the method described above, to serve as standards.