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 containing the bEK coding sequence was kindly provided by Professor Zhao Zhongbao . The plasmids pET28-TEVp5M for expressing the double His6-tagged TEVp5M containing the S219V for inhibiting auto-cleavage, and T17S, N68D, and I77V for enhancing protein solubility, L56V and S135G for improving protein folding, the pET28-GFP for expressing the C-terminal EmGFP to detect N-terminal target protein solubility, the pGST-tevS-eDAL and pCBM-tevS-GFP for expressing the fusion proteins as the TEVp substrates were constructed in our laboratory [12,15]. Since the eDAL is cleaved by the EK, the GST fused to the DAL from Salmonella typhimurium (sDAL) was constructed . MutanBEST Kit for site-directed mutation and reagents used in plasmid construction and protein overexpression were purchased from 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). 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 (L-DAP), o-phenylenediamine (OPA), 2,4-dinitrophneylhydrazine (2,4-DNP) were bought from Sigma (USA).
The C19S, C110S and C130S mutations or their mixed mutations were introduced into the TEVp5M gene in the plasmid pET28-TEVp5M independently and successively using each of the primer pairs C19S1 and C19S2, C110S1 and C110S2, C130S1 and C130S2, and pET28-TEVp5M as the template (Fig. S1). After amplification, the products were phosphorylated, ligated, and sequenced. Then, each of the sequence was excised with Nco I/Xho I and subcloned into Nco I/Sal I sites of pET28-GFP for expressing the constructed TEVp tagged emerald GFP (EmGFP) to quantitatively analyze protein solubility. Due to the Origami (DE3) strain bearing kanamycin resistance, each of the coding sequence through Xba I and Xho I excision was inserted into the Xba I/Xho I sites of the pET-22b vector. In this strain, the genes encoding thioredoxin reductase and glutathione reductase are disrupted for generating the oxidative cytoplasm .
Based on comparison of the mature HRP amino acid sequence (Fig. S1), the sequence encoding the mature mPex Q45-S350 with deletion of the hydrophobic N-terminal leader sequence responsible for directing the protein to the endoplasmic reticulum (ER) 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 placed between the BamH I-Xho I site of pCBM-tevS-GFP to replace the GFP sequence. The similar substitution using the BamH I-Xho I excised bEK coding sequence from the pEK plasmid was conducted for expressing the CBM tagged bEK. The linker between the CBM and the tevS was introduced by amplification of the CBM tag using the primers CBM1 and CBM2, digestion with Nde I and BamH I and insertion into Nde I/Bgl II sites of the pCBM-tevS-GFP plasmids. The flexible linker between the tevS and the bEK was introduced as the similar procedure by using the primers bEK1 and bEK2 for amplification of the bEK coding sequence to prolong sequence upstream of the original BamH I site encoding the extra six-amino-acid, and excising with Bgl II and Xho I for introduction of the BamH I/Xho I sites of the former constructed plasmids. The BamH I-Xho I excised fragment encoding bEK was then substituted with that encoding the mPex.
For constructing the redox sensitive GFP reporter roGFP , the sequence encoding the GFP variant mGFP5 with improved stability in E. coli , was ampliﬁed using the plant expression vector pCAMBIA1302 as the template and primers mGFP5-1 and mGFP5. The purified PCR amplicon with the Nco I and Hind III was subcloned into the Nco I-Hind III site of the pET-28b plasmid. The mutations C48S, S147C and Q204C were introduced into the mGFP5 step by step by using the primers for PCR. All inserts were sequenced to identify correction. The primers used in this study were listed in the table S1.
Solubility determination of the overexpressed TEVp constructs in different E. coli strains
Except where noted, induction and extraction of recombinant proteins in this study were conducted as follows. The constructed plasmids were transformed independently into the different E. coli strains. The recombinant cells were cultured overnight at 37 °C in 5 ml of Luria-Bertani (LB) broth, diluted to 50-fold and grown at 37°C. Induction of the target protein was performed by addition of isopropylthio-β-D-galactoside (IPTG) at final concentration 0.5 mM, when OD600 value reached about 0.5. After cultured at 28 °C for 12 h in 10 ml liquid culture of a 50-ml shake flask, cells were collected by centrifugation, washed with buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), sonicated and centrifuged, and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein amounts were measured by Bradford method, using bovine serum albumin as the reference. After electrophoresis, proteins on the gel were transferred to polyvinylidene ﬂuoride membrane, immunoblotted with anti-His6 monoclonal antibodies, and treated with HRP conjugated anti-rabbit 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.
The C-terminally fused EmGFP reporter is 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 .
Purification of the constructed TEVp with C110S and/or C130S mutations
The expression plasmids were transformed into the Rossetta (DE3) strain with supply rare tRNAs for augmenting soluble expression level of the TEVp construct. After induction at 28 °C for 12 h, cells 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. The supernatants were loaded on a Ni-NTA spin, centrifuged twice with buffer B, washed twice with 40 mM imidazole in buffer B (pH 8.0), and eluted with 250 mM imidazole in buffer B (pH 8.0). Purified protein was concentrated by an Ultra-15 centrifugal filter tube equipped with an Ultracel-10 membrane, and exchanged with buffer A. The freshly prepared protein was used for cysteine labeling and activity assay.
Modification of the free cysteine residues in the purified TEVp constructs
The free cysteine residues of the TEVp constructs were labeled with AMS as the described method . Purified TEVp variants was incubated with 150 µM CuCl2 as the oxidative agent or 1 mM dithiothreitol (DTT) for 1 h at 25 °C, precipitated by trichloroacetic acid. After it was washed and re-suspended with buffer A twice, the precipitated protein was labeled with AMS, a maleimidyl reagent specifically alkylating free SH-group of cysteine to increases the molecular weight in about 0.5 kDa. When the reaction was finished, the mixture was centrifuged and washed with buffer A. The labeled TEVp variants were incubated with loading buffer in absence of DTT, and separated by 12% SDS-PAGE under non-reducing condition.
Quantitative analysis of in vitro activity
In vitro cleavage activity was assayed using the GST-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 in the buffer A. The cleavage was reacted at 30 °C for 1 h, and the activity was determined by coupled assay of the eDAL activity . As a PLP dependent enzyme, eDAL catalyzes L-DAP to pyruvate and ammonia. The amount of pyruvate was measured with 2,4-DNP. The reaction mixture for testing DAL activity contained 50 μM PLP and 10 mM L-DAP, and the crude extracts 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, absorbance at 520 nm representing the amounts of pyruvate in the mixture was measured on a U-2001 spectrometer (Hitachi, Japan).
Recovery of inclusion bodies
The induced cells were re-suspended in buffer A and disrupted by sonication. After centrifugation at 4000 g for 10 min, insoluble fraction was collected, washed with buffer A twice and re-suspended with buffer B [30 mM Tris/HCl, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, pH 7.5]. The mixture was centrifuged and IBs were re-suspended with buffer B in the absence of Triton X-100.
The washed IBs containing the bEK was solubilized in buffer A containing 8 M urea, 5 mM DTT (V/V) and 10 mM EDTA . The mixture was left for 2 h at room temperature to ensure sufficient amounts of IBs for solubilization and reduction of the mismatched disulfide bonds, and centrifuged at 18000 g for 30 min to remove the pellet. IBs containing the mPex dissolved in the solution (4.5 M urea, 40 mM Tris-HCl, pH 9.0, 5 mM DTT and 0.2 mM hemin) to a protein concentration of 0.3 mg/mL, with slight modification . The resultant suspension was centrifuged at 12000 g for 20 min and the debris was discarded. The solubilized protein was used for refolding.
Matrix-assisted refolding of target proteins
For increasing the cellulose loading yields of target proteins, the regenerated amorphous cellulose (RAC) was prepared based on the published paper . The microcrystalline cellulose was suspended with double-distilled water at ratio of 1:3 (W/V), and slowly added 20 fold volume of 10 ml ice-cold H3PO4 with vigorous stirring, than supplemented 2 fold volume of ice-cold water. After centrifugation, the pellet was suspended with ice-cold water several times for removing phosphoric acid in soluble fraction, and centrifuged. With adding 2 M Na2CO3 to the pellet, the mixture was centrifuged and discarded. After the resin was washed with ice-cold water several times until the pH value in mixture containing RAC was reached about 7.0. The solubilized proteins from IBs were mixed with RAC at mass ratio of about 1:20 to make the urea concentration decreased from 8 M to 6 M. The mixture was stirred for 1 h at room temperature to allow the target protein binding RAC.
For the bEK refolding , the protein was diluted with buffer C (100 mM Tris–HCl, 6 M urea, 10 mM cystine, pH 8.0) to facilitate correct formation of disulﬁde bonds. Then, the resin was put in, mixed in buffer C containing 3 M urea 5% glycerol (V/V) and 10 mM cysteine, and diluted with buffer C containing 0.7 M urea, 15% (v/v) glycerol, 0.5 mM cysteine-HCl, 5 mM cysteine and 2mM CaCl2. For the mPex refolding with slight modification , the denatured proteins were diluted with buffer D [20 mM Tris-HCl, pH 8.5, containing 0.5 M urea, 5% glycerol (V/V), 2 µM hemin, 2 mM CaCl2, 0.5 mM GSH and 5 mM GSSG] in the presence of RAC.
Purification and activity assay of the refolded tag-free proteins by the TEVp variant digestion
The purified TEVp variant was incubated with the refolded protein bound to the RAC with mass ratio of 1: 10 at 10 °C for 24 h. After reaction was finished, the Ni-NTA-resin was added and incubated for 2 h at room temperature. The supernatant after centrifugation was subjected to SDS-PAGE analysis. The bEK cleaving the GST tagged sDAL with incorporation of D4K as the bEK recognition sequence was analyzed, based on the coupled assay of sDAL activity. 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 mixture (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.