Plasmids, host strain, and reagents
The abbreviations of all proteins used in this work and gene accession numbers were listed in Table 1. E. coli strains DH5α and BL21 (DE3), and the plasmid pET-22b and pET-28b are products of Novagen (USA). B. subtilis strain 168 and its expression plasmid pTH43 were kindly provided by Dr. Mu [23]. Human cDNA library was provided by Maikun Teng, University of Science and Technology of China. The genes as BamH I-Xho I excised fragment encoding the proteins were constructed previously. The genes encoding the EmGFP, mCherry, tobacco etch virus protein variant termed as TEVp5M, ScUlp1, mSR and the mature mSF (V71-S212), and the codon-optimized Vhb with insertion at BamH I and Xho I sites in the expression vectors, and the helper plasmid for expressing yeast mature 5-aminolevulinic acid synthase (ALAS), the plasmid for expressing the SUMO-EmGFP as the ScUlp1 substrate, GST-EmGFP as the TEVp5M substrate were constructed, and the tag-free Vhb was purified in our laboratory [24-26]. The bacteriophage ΦX174 gene E encoding the lytic protein (LyE) was inserted into the pBAD33.1 plasmid for controlling the gene expression under the PBAD promoter, and the cumate inducible promoter was inserted into the pET-22b to yield Pcum-22b plasmid [27]. The secretion expression plasmid pPICZαA, P. pastoris strain KM71H and EasySelect Pichia Expression Kit are products of Invitrogen, USA.
Heparin Sepharose CL-6B, Phenyl Sepharose CL-4B and amylose resin were obtained from GE (Healthcare, USA). Nickel-nitrilotriacetic acid (Ni-NTA) agarose was purchased from Qiagen (Hilden, Germany). Ultra-15 centrifugal filter tube equipped with the Ultracel-10 membrane, 3-hydroxyanthranilic acid (HAA), Zeocin, D-serine (D-Ser), 5-aminolevulinic acid (ALA), pyridoxal 5’-phosphate (PLP), o-phenylenediamine (OPA), 2,4-dinitrophneylhydrazine (2,4-DNP) were purchased from Merck-Sigma-Aldrich (Kenilworth, NJ, USA). Primers and gene were synthesized in General Biol Company (Chuzhou, Anhui, China). Antibodies and reagents for Western blotting were bought from GenScript Company (Nanjing, Jiangsu, China). Trypsin and its inhibitor were purchased from Sangon Biotech (Shanghai, China).
Plasmids construction
The sequence encoding hanA1 was amplified using human brain cDNA library as template and primers P1 and P2. The gel-purified PCR products were digested with Nde I and BamH I, and inserted into Nde I/Bgl II sites of the our constructed pCBM-GFP plasmid [24], to generate the resultant vector pA-EmGFP for expressing hanA1 tagged EmGFP with the incorporation of tobacco etch virus protease (TEVp) recognition sequence ENLYFQG (tevS), or into Nde I/BamH I sites of the pET-28b plasmid (Novagen, USA) to express the His6-tagged hanA1-EmGFP. The coding sequence was also amplified by using primers P1 and P3. With incubation of Xba I and Nhe I, the fragment was inserted into Xba I site of the pTH43 plasmid with Xba I excision followed by E. coli alkaline phosphatase digestion to express the hanA1-EmGFP, or mCherry. This plasmid is a shuttle vector harboring the resistance to ampicillin in E. coli, and carrying the chloramphenicol resistant gene for selecting B. subtilis transformants. To overexpress the hanA1-EmGFP in P. pastoris, the PCR-amplified sequence encoding the hanA1-EmGFP with primers P1 and P3 was incubated with EcoR I and Nhe I, and inserted into EcoR I and Xba I sites of the pPICZαA plasmid (Invitrogen, USA) for expressing the target protein fused with the α factor signal peptide controlled by the PAOX promoter.
The open reading frame (ORF) of the Bacillus cereus phospholipase C (BcPLC) was amplified using primers P4 and P5 to subclone the Nde I and Hind III sites of the Pcum-22b plasmid. The ORF of hS100A11 was amplified by using the cDNA library as the template, with primers P6 and P7. The PCR products were treated with BamH I and Xho I, and inserted into the BamH I-Xho I site of the pCBM-GFP plasmid for expressing the codon optimized cellulose-binding module (CBM) tagged hS100A11, or into the same sites of the pMBP-eDAL plasmid [25] to produce the MBP-hS100A11 for immobilizing the protein in the affinity matrix. The EcALAD coding sequence was amplified using the E. coli genomic DNA as the template, and the primer pairs P8 and P9, treated with BamH I and Xho I and subcloned into BamH I/Xho I sites of the pA-EmGFP plasmid. The fragment encoding ScHAD was amplified by using Saccharomyces cerevisiae genomic DNA as the template, and primers P10 and P11. With BamH I and Xho I treatment, the amplicon was subcloned downstream of the hanA1 coding sequence. The gene encoding the EcLDC was also amplified by PCR using E. coli genomic DNA as the template and primers P12 and P13, and amplified products were subcloned into BamH I-Xho I sit of the pA-EmGFP to overexpress the hanA1-EcLDC. The coding sequence was also subcloned into the pET-28b to produce His6-tagged hanA1-EcLDC. The fragment encoding the codon variant of the mature mCAO (S39-R708) was synthesized inserted into the BamH I-Xho I site of the pA-EmGFP plasmid to express the hanA1-mCAO, based on the amino acid sequence comparison of the other plant homologue for soluble production in E. coli [28]. The codon optimized CrLOV was also constructed as the same procedure, based on the presented amino acid sequence [29]. Each of the genes was substituted for the EmGFP coding sequence at the BamH I and Xho I sites. All constructs were sequenced to identify the insert correction.
Detection and separation of the hanA1 tagged EmGFP in E. coli cytoplasm
The transformed E. coli BL21(DE3) cells were cultured overnight at 37 °C in lysogeny broth (LB, yeast extract 5 g/L, tryptone 10 g/L, NaCl, 10 g/L) with supply of 40 μg/mL kanamycin and diluted to 200 folds. When optical density at 600 nm (OD600) of cells was reached about 0.5 as measured on a U-2900 ultraviolet-visible spectrometer (Hitachi, Tokyo, Japan), the fusion protein was induced by use of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 28 °C for 12 h. After centrifugation, cells were re-suspended in buffer A (20 mM Tris-HCl, pH 8.0, 10% glycerol), and photographed under the fluorescent microscope (Olympus, Japan) with the excitation peak 488 nm.
Cells were washed with buffer A, and disrupted by sonication at 4°C. After centrifugation at 13000 g for 20 min, the target protein was precipitated by addition of CaCl2 at different concentrations. The precipitant was dissolved in 10 ml buffer A containing EDTA-Na2 at higher concentrations over CaCl2, shaken at 100 rpm for 30 min, and centrifuged. The supernatant was concentrated with Ultra-15 centrifugal filter tube and exchanged with buffer A. The fluorescence signal was measured with excitation and emission wavelengths at 488 nm and 509 nm in a F-4500 fluorescence spectrometer (Hitachi, Japan). For quantifying the hanA1-EmGFP in each purification step, recombinant cells were cultured in 20 ml medium in 50 ml flasks. The purification table was prepared based on the GFP fluorescence, according to the published report [30].
Detection and separation of the hanA1 tagged proteins in the culture
The recombinant E. coli cells were induced to overexpress the hanA1-EmGFP at 37°C for 5 h, then, 13 mM arabinose was added for 1 h to induce the LyE production for causing the cell lysis. After centrifugation, cells were collected and photographed under the fluorescent microscope. Different concentrations of CaCl2 were added to the supernatant, and the precipitants were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein secretion was also conducted by pre-overexpression of the BcPLC with 50 μM cumate induction at 28°C for 6 h [27], and addition 0.5% Triton X-100 to the LB medium. The target protein was induced by use of 0.4 mM IPTG at 28°C for 12 h, based on the former study [31]. With removal of cells, CaCl2 at final concentrations of 20, 50 and 100 mM to the culture, and centrifuged, the precipitated proteins were detected by SDS-PAGE.
The electro-transformation of plasmids into B. subtilis 168 strain cultured in LB containing 5 μg/ml chloramphenicol in the presence of trehalose is performed, according to the described method [32]. The transformed B. subtilis cells were cultured overnight at 37°C. With dilution to 100 folds, the cells were grown until OD600 reached about 0.5. With use of 0.05 mM IPTG for induction and 0.07% Triton X-100 to the culture, cells grew for another 48 h and were photographed. The target protein was prepared by 20, 50 and 100 mM CaCl2 mediating precipitation.
The plasmids pPICZα-hanA1-EmGFP were linearized by Sac I excision and transformed into the strain KM71H by electroporation. The colonies were inoculated on an YPDS (1% yeast extract, 2% peptone, and 2% dextrose) plate containing 1 mg/mL Zeocin for selecting multicopy integrated strains. The selected strains were inoculated in 500 mL of BMGY medium [1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% yeast nitrogen base (YNB), 4×10-5 % biotin, 1% glycerol] in 1-L Erlenmeyer flasks and cultivated at 30°C and 250 rpm for 24 h. The cells were recovered by centrifugation at 2,000 g for 10 min at room temperature and then re-suspended in 100 mL BMMY medium in 1-L Erlenmeyer flasks. The media were then incubated at 30°C for 6 days in a shaker at 250 rpmin in the presence of 0.5% Triton X-100. To maintain induction, 0.5 mL of 100% methanol was added every day. The collected cells were photographed. The protein via extracellular production was treated with 20, 50 and 100 mM CaCl2, and insoluble fractions were analyzed by SDS-PAGE.
Protein analysis
Protein concentration was analyzed by Bradford method using bovine serum albumin as the reference. Proteins samples were analyzed by SDS-PAGE. For Western blot analysis, the fusion protein was analyzed by SDS-PAGE and transferred to polyvinylidene fluoride membrane, immunoblotted, and treated with the primary antibody and horseradish peroxidase conjugated secondary antibodies. The bands were appeared and photographed by adding 0.08% hydrogen peroxide and 4-chloro-1-naphthol solution (dissolved in 20% methanol). For observation of the fusion protein for the fluorescence on the gel, protein samples were mixed with SDS-PAGE sample buffer, incubated at 45°C for 10 minutes, centrifuged briefly and separated. The gel was later visualized under UV light and photographs documented [25].
Column-dependent affinity purification and on-resin cleavage
The hanA1-EmGFP from soluble extracts or affinity separation was dissolved in buffer A containing 1 mM CaCl2 (Buffer B), further purified by heparin Sepharose CL-6B (1.0×5 cm), based on the published report [16]. The column was washed with buffer B, and the fusion protein was eluted with buffer A containing 5 mM heparin. For purifying the fusion protein by phenyl Sepharose CL-6B (1.0×3 cm) at room temperature [16], the separated fusion protein was loaded on the resin for stepwise elution with 30-0% saturated ammonium sulfate. The eluted protein was analyzed by SDS-PAGE.
For oriented immobilization of the MBP-hS100A11, amylose resin was washed with buffer A containing 250 mM NaCl, followed with buffer B, the bound hS100 was used for absorbing with the hanA1-EmGFP in crude extracts or via affinity separation. With washing with buffer B, the target protein was eluted with buffer A containing 5 mM EDTA-Na2 (Buffer C). To enhance immobilization of the hS100A11, the constructed CBM-hS100A11 was strongly bound to the regenerated amorphous cellulose (RAC), based on the published method [24]. After incubation at room temperature for 3 h, the resin was washed three times with buffer A containing 500 mM NaCl for removal of the contaminated proteins, and re-suspended with buffer B. The hanA1-EmGFP dissolved in buffer B in clear lysate or via affinity separation was incubated with the immobilized hS100A11 at room temperature for 2 h. The resin was washed with buffer B containing 50 mM NaCl, and the target protein was eluted with buffer C. On-resin cleavage was finished at room temperature for 5 h by using purified His6-tagged TEVp5M at mass ratio of 1:5 for TEVp5M and the bound proteins in buffer B. The hanA1-EmGFP via soluble extraction or affinity separation was dissolved in buffer B, and incubated phosphatidylserine at room temperature for 12 h. Then, the proteins were washed with buffer B, and target protein was eluted with buffer C.
The His6-tagged hanA1-EmGFP or mSF was loaded on Ni-NTA resin with buffer D (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0), washed with buffer D containing 30 mM imidazole (pH 8.0), and eluted with buffer D containing 200 mM imidazole (pH 8.0).
Visualization of affinity separation process using different colored proteins
The tagged EmGFP, mCherry, CrLOV, and mSF were produced as the above-mentioned inductions. Coexpression of ScALAS increases the holo-Vhb in E. coli [24]. The supernatants were added CaCl2 at final concentration of 10 mM, and re-solubilized protein with supplemented 15 mM EDTA-Na2 (final concentration). The separated and solubilized proteins were photographed under UV or visible light. The His6-tagged mSF was purified by Ni-NTA, and the eluted protein in buffer A was analyzed on the spectrometer for recording UV–Vis absorption spectra at 300–700 nm, and on a Bruker ELEXSYS E500 spectrometer for recording electron paramagnetic resonance (EPR) spectra. EDTA-Na2 was added to solubilize His6-tagged mSF, and the chelating affect was measured based on change at A415 [33]. The chelating agent was also added to the prepared solution containing the heme-bound Vhb, and A405 was measured [34].
Activities analyses of different enzymes
For detecting the tagged TEVp5M and ScUlp1 cleavage activities, two sets of the fusion protein were constructed. The fused EmGFP constructs were constructed for detecting the cleavage activities, based on the in-gel fluorescence display. Cleavage of the fusion tag increases significantly increased E. coli diaminopropionate ammonia-lyase (EcDAL) activity, which is measured at absorption at 520 nm to indicate pyruvate amounts in reaction mixture [35]. The fusion protein for the EcDAL catalyzing the pyruvate generation was used as control and A520 was subtracted. One unit of enzyme activity was defined as the amount of enzyme required to catalyze the release of 1 μmol of pyruvate/min at 37°C, pH 8.0. The mSR catalysis for eliminating D-Ser to generate pyruvate with 2,4-DNP was measured [36]. The mixture in 0.5 mL contained 20 mM Tris/HCl, pH 8.5, 10 μM PLP, 10 mM D-Ser, 1 mM CaCl2, and freshly separated mSR in the presence or absence of 10 mM EDTA-Na2. The reaction occurred at 37 °C for 30 min, stopped by adding 0.5 mL of 2 M HCl containing 0.03 % (w/v) DNP. Following by centrifugation and incubation at 4°C for 5 min, 1 mL of 2 M NaOH was added, the mixture was centrifuged, and absorbance at 520 nm (A520) representing pyruvate amounts in the supernatant was measured. The assay for the tagged EcALAD was analyzed for measuring amounts of porphobilinogen as the catalytic product [37]. In 0.5 mL mixture, 50 mM of potassium phosphate buffer (pH7.5), 50 μM of ZnCl2, 1 mM of MgCl2, 5 mM of ALA, and freshly separated EcALAD in the presence or absence of 10 mM EDTA-Na2 were added for reacting at 37°C for 10 min. The amounts of porphobilinogen were determined with addition of same volume of freshly prepared Ehrlich's reagent (0.2 g p-dimethylaminobenzaldehyde dissolved in 2.5 mL HCl, mixed with 7.5 mL glacial acetic acid). The mixture was centrifuged and absorbance at 555 nm for the supernatant was measured. ScHAO activity was measured the formed 2-amino-3-carboxymuconate-6- semialdehyde (ACMS). In 1 ml mixture, 50 μM MOPS buffer, pH 7.0, 0.1 μmol 3-HAA, 0.1 μmol Fe(NH4)2(SO4)2 · 6H2O, and the separated separated ScHAD with or without EDTA-Na2, were added and incubated at 25°C for 3 min, A360 was measured [38]. The mCAO activity was measured by the coupled assay. Cadaverine was oxidized by the recombinant mCAO to yield hydrogen peroxide, which is formed by HRP and pigmented by OPA. The absorption at 411 nm was assayed [39].
Cross-linking of the separated hanA1 tagged EcLDC
For increasing purity of the target protein in cross-linked enzyme aggregates (CLEA), the hanA1 tagged EcLDC was induced in E. coli at 16°C for 24 h, precipitated by buffer A containing 10 mM CaCl2, washed with the same buffer to remove impurities, and re-suspended. Then, glutaraldehyde was added into the mixture to a final concentration of 25 mM with slowly stirring for 20 h at 4°C to crosslink the aggregated proteins [40]. After centrifugation at 12,000 g for 10 min, the pellet was washed twice with potassium phosphate buffer (100 mM, pH6.0) to remove the unreacted glutaraldehyde and re-suspended with the same buffer. The prepared EcLDC aggregates (hanA1-EcLDCCLEA) were stored at 4°C. The trypsin digestion, leaky amounts and reusability of the hanA1-EcLDCCLEA were analyzed.