Bacterial Strains, Plasmids and Reagents
E. coli strains DH5α, BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3), the plasmids pET-22b and pET-28b are products of Novagen (Madison, WI). The plasmid pET32a-pEK was kindly provided by Professor Zhao Zhongbao . The plasmids for expressing the double His6-tagged TEVp5M, the emerald green fluorescent protein (EmGFP) as the C-terminal solubility reporter, the GST-tevS-eDAL, the CBM tag fused to the linker GGTGGS around the tevS, and the GST-tevS-sDAL were constructed in our laboratory [8,17,20]. MutanBEST Kit for site-directed mutation and reagents for plasmid construction and protein overexpression were supplied by Takara (Dalian, China). Nickel-nitrilotriacetic acid (Ni-NTA) agarose was made by Qiagen (Chatsworth, CA). Ultra-15 centrifugal ﬁlter tube equipped with Ultracel-10 membrane was obtained from Amicon (USA). Mouse anti-His6 monoclonal antibody and horseradish peroxidase (HRP) conjugated anti-mouse IgG antibody were obtained from GenScript, China. The compound 4-acetoamide-4’-maleimidyl-stilbene-2,2’- disulfonate (AMS) was purchased from Invitrogen (USA). The compounds including pyridoxal 5’-phosphate (PLP), DL-α, β-diaminopropionate (DL-DAP), o-phenylenediamine (OPA), 2,4-dinitrophneylhydrazine (2,4-DNP) were bought from Sigma (USA).
Each and combined mutations C19S, C110S and C130S were introduced into the TEVp5M using the primer pairs C19S1 and C19S2, C110S1 and C110S2, C130S1 and C130S2 (Tab. S1), and pET28-TEVp5M as the template. After amplification, the PCR products were phosphorylated, ligated, and sequenced. The sequence encoding the TEVp variant was excised with Nco I/Xho I and subcloned into Nco I/Sal I sites of pET28-GFP for expressing the TEVp variant fused to the EmGFP. Since the Origami (DE3) strain bears kanamycin resistance, the tagged TEVp coding sequence with Xba I and Xho I excision was inserted into the Xba I/Xho I sites of the pET-22b vector.
According to comparison of the mature HRP amino acid sequence (Fig. S1), the fragment encoding the mature mPex Q45-S350 was amplified by RT-PCR using the total RNAs extracted from maize leaves as the template, and primers mPex1 and mPex2. The PCR amplicon was incubated with BamH I and Xho I, and inserted into the BamH I-Xho I site of the plasmid encoding the CBM tag. The plasmid for expressing the CBM tagged bEK was constructed as the same procedure.
Production of the TEVp variants in different E. coli strains
Except where noted, induction and extraction of recombinant proteins in this study were conducted as follows. The plasmids were transformed into the E. coli strain. The recombinant cells were cultured overnight at 37 °C in 5 ml of lysogeny broth (LB), diluted to 50-fold and grown at 37°C. When OD600 value was reached about 0.5, as measured on a U-2900 spectrometer (Hitachi, Japan), the target protein was induced by use of 0.5 mM isopropylthio-β-D-galactoside (IPTG). After cultured at 28 °C for 12 h in 10 ml liquid culture of a 50-ml shake flask at 220 rpm, cells were collected by centrifugation (4000 g, 10 min, 25 °C), washed with buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), sonicated with 3 s for 198 times, and 10 s interval at 4 °C. Followed by centrifugation (12000 g, 15 min, 4 °C), the soluble and insoluble fractions were collected, and the pellet was washed twice with buffer A, and dissolved in 8 M urea, and centrifuged (12000 g, 15 min, 25 °C) to remove the precipitant. Protein amounts were determined by Bradford method, using bovine serum albumin as the reference. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins on the gel were transferred to polyvinylidene ﬂuoride membrane, immunoblotted with anti-His6 monoclonal antibodies, and treated with affinity purified HRP-conjugated goat anti-mouse IgG. The band representing the target protein was visualized by addition of 4-chloro-1-naphthol solution dissolved in 20% methanol and 0.08% H2O2.
Solubility Analysis of the TEVp Variants
The C-terminally fused EmGFP reporter was used for quantitative analysis of the soluble TEVp amounts, based on the fluorescence emitted from soluble fractions on the F-4500 fluorescence spectrometer (Hitachi, Japan) with excitation and emission maximum 488 and 515 nm .
Coupled Assay of the Cleavage Activity
In vitro cleavage activity was assayed using the GST-tevS-eDAL purified by Ni-NTA . The mass ratio was 30:1 for the purified protein substrate and soluble extracts containing the recombinant TEVp construct, and 50:1 for purified protein substrate and the protease. The cleavage was reacted at 30 °C for 1 h, and the activity was determined by coupled assay. This enzyme depends on the PLP cofactor and catalyzes DL-DAP to pyruvate and ammonia. The reaction mixture for testing eDAL activity contained 50 μM PLP and 10 mM DL-DAP, and the prepared eDAL in a final volume of 1 ml. After incubated at 37 °C for 5 min, 1 ml of 2 mM HCl plus 0.03% 2,4-DNP was added to stop the DAL catalytic reaction. Following incubation at 4 °C for 5 min, 2 ml of 2 M NaOH was supplemented. After centrifugation (12000 g, 10 min, 25 °C), absorbance at 520 nm representing pyruvate amounts was measured.
Purification of the Soluble TEVp Variants
The expression plasmids were transformed into the Rossetta (DE3) cells. After induction at 28 °C for 12 h, cells in 500 ml LB culture were collected by centrifugation and washed with buffer B (50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole, pH 8.0), and disrupted by sonication with 3 s for 198 times, and 10 s interval at 4 °C. The supernatant was loaded on a column containing 4 ml Ni-NTA with pre-equilibration with 40 ml buffer B, washed twice with 40 ml in buffer B (pH 8.0) containing 40 mM imidazole, and eluted with 40 ml buffer B (pH 8.0) containing 250 mM imidazole. Purified protein was concentrated by use of the Ultra-15 centrifugal filter tube equipped with the Ultracel-10 membrane, and exchanged with buffer A. Purity of the TEVp variants were characterized by SDS-PAGE.
Modification of Free Cysteine Residues in Purified TEVp proteins
The free cysteine residues of the TEVp constructs were labeled with AMS as the described method . Purified TEVp variants was incubated with either 150 µM CuCl2 for oxidizing the free cysteine residues on the TEVp surface or 1 mM dithiothreitol (DTT) for reducing the oxidized cysteine ones at 25 °C for 1 h, and precipitated by trichloroacetic acid. After it was washed twice, centrifuged (12000 g, 1 min, 25 °C) and re-suspended with buffer A, the precipitated protein was labeled with AMS, a maleimidyl reagent specifically alkylating free thiol group of cysteine to increase molecular weight up to 0.5 kDa . When the reaction was lasted for 20 min at room temperature, the mixture was centrifuged (12000 g, 10 min, 4 °C) and washed with buffer A. The labeled TEVp variants were incubated with the SDS-PAGE loading buffer in absence of DTT at 100 °C for 10 min, and separated by 12% SDS-PAGE under non-reducing condition.
Refolding of the Fusion Proteins and Release of the Target Enzymes via Tag Removal
The IBs from the BL21(DE3) cells carrying the plasmids encoding the CBM tagged bEK or mPex were collected, washed and re-suspended with buffer C [30 mM Tris/HCl, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, pH 7.5]. Then, IBs were re-suspended with buffer C in the absence of Triton X-100. The tagged bEK in the prepared IBs was solubilized with buffer D (30 mM Tris-HCl, 200 mM NaCl, 8 M urea, 5 mM DTT, and 10 mM EDTA-Na2). To ensure sufficient amounts of IBs to be solubilized, and the mismatched disulfide bonds in the proteins were reduced, the mixture was incubated at room temperature for 2 h and centrifuged (18000 g, 30 min, 25 °C) to remove the pellet . The mPex in the IBs was solubilized with buffer E (40 mM Tris-HCl, pH 9.0, 4.5 M urea, 5 mM DTT) to a protein concentration of 0.3 mg/mL, according to the published report with slight modification . The suspension was centrifuged (18000 g, 30 min, 25 °C) and the solubilized protein was collected.
For increasing more amounts of the CBM tag than the cellulose, RAC was prepared, as previous described procedure . For the bEK refolding, the protein was diluted with buffer F (100 mM Tris–HCl, 6 M urea, 10 mM cystine, pH 8.0), and RAC was added. The mixture was diluted slowly with buffer G [80 mM Tris–HCl, 0.7 M urea, 15% (v/v) glycerol, 0.5 mM cysteine, 5 mM cysteine, 2 mM CaCl2]. For the mPex refolding, the denatured proteins were diluted with buffer H [40 mM Tris-HCl, pH 8.5, 0.5 M urea, 5% glycerol (V/V), 2 µM hemin, 2 mM CaCl2, 0.5 mM GSH and 5 mM GSSG]. After the refolding process was finished, the mixture was centrifuged (3000 g, 10 min, 25 °C), the resin was washed three times with buffer G for the bound bEK, or four times with buffer H in absence of hemin for the bound mPex. Purified TEVp C110S/C130S variant was incubated with the refolded protein bound to RAC with mass ratio of 1: 10 at 10 °C for 24 h. Then, Ni-NTA resin was added and incubated for 2 h at room temperature. Followed by centrifugation (3000 g, 10 min, 25 °C), the supernatant was collected.
Activity Assay of the Refolded Enzymes
The prepared protein samples were subjected to SDS-PAGE analysis. The bEK cleaving the GST tagged sDAL with incorporation of the D4K as the bEK recognition sequence was analyzed, based on the coupled assay of the sDAL activity. The bEK can cleave the eDAL at the undesired site to inactivate the eDAL . The mPex catalyzes the degradation of H2O2 using OPA as a hydrogen donor, which turns yellow upon oxidation . The freshly prepared mPex was incubated in the buffer I (20 mM Tris-HCl, pH 7.5, 50 μg/mL OPA, 10 mM or 30 mM H2O2) at 37 °C for 30 min, and absorption at 411 nm was measured.
For solubility analysis, five samples from different colonies randomly selected for culturing were conducted and the data from the closest value of three biological replicates were calculated. For activity assay, the data were from three technique replicates. Data were indicated as means ± standard deviations (SD). Data were evaluated using a one-tailed t-test. The data were analyzed using SPSS ver. 22 (SPSS Inc., USA).