After centrifuging the cultures at 4 °C with 9000 rpm for 20 min, the supernatant was loaded onto a Ni-NTA sepharose column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and performed purification process with AKTA150 automatic purification system. Imidazole of gradient concentration (25–500 mM) in 20 mM phosphate buffer (pH 7.6, 500 mM NaCl) was used for the elution of target protein. The molecular weight and purity of the purified CsnB were assayed by SDS-PAGE on a 12% (w/v) resolving gel was utilized to measure the concentration of CsnB using bovine serum albumin (BSA) as standard. The optimal temperature of CsnB was determined at different temperatures (0–80 °C). To measure the thermo-stability of CsnB, the purified CsnB was firstly incubated at various temperatures (0–100 °C) for 30 min and 60 min, respectively. Then, the enzymatic activity was determined at its optimal condition. To determine its optimal pH, the enzyme was fixed with different buffer as follows: HAC-NaAC, pH 3.71–5.76; phosphate buffer, pH 5.89–7.29; Tris-HCl buffer, pH 6.45–7.78; and glycine-NaOH buffer, pH 6.15–8.67. Moreover, the pH-stability of CsnB was determined at its optimal condition after the enzymes incubated at different buffers [HAC-NaAC (pH 3.58–5.58); phosphate buffer (pH 6.97-8.10); Tris-HCl buffer (pH 7.37–8.60); glycine-NaOH buffer (pH 8.78–9.90)] at 4 °C for 24 h.
Ni nta sepharose column
Manufactured by GE Healthcare
Sourced in United States, Sweden
The Ni-NTA Sepharose column is a chromatography resin designed for the purification of His-tagged proteins. It consists of nickel-nitrilotriacetic acid (Ni-NTA) coupled to Sepharose beads, which allows for the specific and reversible binding of His-tagged proteins. The column can be used in a variety of protein purification workflows.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protein quantitative analysis kit, by Beyotime (2 mentions)
Coomassie brilliant blue g 250, by Beyotime (1 mentions)
Lb media, by Merck Group (1 mentions)
Bradford assay kit, by Sangon (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Ovalbumin (ova), by GE Healthcare (1 mentions)
Hiload 16 600 superdex 200 prep grade column, by GE Healthcare (1 mentions)
Nanophotometer n60, by Implen (1 mentions)
14 protocols using ni nta sepharose column
1
Purification and Characterization of CsnB
2
Expression and Purification of vNAR Proteins
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The vNAR genes from the chosen clones were inserted into the pcoldII plasmid and chemically transformed into Shuffle T7 competent E. coli (NEB) using CaCl2. The strains containing recombinant vNARs were grown for protein expression in 2 YT with 100 mM of ampicillin until the OD600 reached nearly 0.8. Protein production was induced with 0.5 mM IPTG by shaking for 20 hat 16 °C. The collected bacteria were sonicated via ultrasound and proteins were initially purified on the Ni-NTA Sepharose column (GE Healthcare) followed by SEC. For proton influx and patch clamp functional assays, the gene was inserted into the pcold-sumo vector to avoid the effect of His-tag. The sumo tag with 6×His was cleaved by sumo enzyme and tag-cleaved vNARs were purified on a second Ni-NTA Sepharose column. The eluate from Ni-column was run on SEC using Superdex 75 column for further purification. The eluted fractions were evaluated by SDS-PAGE for purity and concentrated using concentrators with a molecular mass cut-off of 3 kDa. The concentration was quantified using a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific Inc.).
3
Recombinant Alginate Lyase Production in Yarrowia lipolytica
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The ALYW202 gene with the XPR2 signal peptide gene was synthesized after codon optimization (Synbio Technologies, Suzhou, China). The synthesized alginate lyase gene was ligated into expression vector pINA1312 and then transformed into URA- strain [41 (link)]. The recombinant Y. lipolytica URA- harboring the pINA1312/ALYW202- XPR2 was cultured in a GPPB liquid medium for 84 h cultivation at 180 rpm and 30 °C. The recombinant strain Y7 was chosen for the highest extracellular activity. During the fermentation of Y7 in flasks, the alginate lyase activity and the biomass were detected every 12 h. All the data were collected in triplicate. The supernatant of strain Y7 containing the recombinant protein was loaded onto a Ni-NTA sepharose column (GE Healthcare, Chicago, IL, USA) equilibrated with lysis buffer (pH 7.0) as mentioned before. The binding buffer (50 mM phosphate buffer, pH 7.0, 500 mM NaCl, 20 mM imidazole, pH 7.0) was used to equilibrate the resin, while the elution buffer (50 mM phosphate buffer pH 7.0, 500 mM NaCl, 500 mM imidazole, pH 7.0) was used to obtain the intracellular protein. The active fraction was analyzed by 12% (w/v) SDS-PAGE gel.
4
Production and Purification of Alginate Lyase
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The E. coli cells harboring the recombinant plasmid pET32a-algB were cultured at 37 °C in liquid LB medium with 100 μg/mL ampicillin until OD600 reached about 0.6. Gene expression was induced with the addition of isopropyl-β-d -thiogalactoside (IPTG) to a final concentration of 0.8 mmol/L and then grown at 20 °C for 24 h. The cells were harvested by centrifugation 8000 r/min, 5 min, at 4 °C, then suspended in 0.05 mol/L Tris-HCl buffer (pH 7.4) to concentrate 1/10 volume. The cells were disrupted by ultrasonication. The lysate was harvested by centrifugation 10,000× g, 30 min, at 4 °C. The supernatant was filtered with 0.22 micrometer cellulose acetate membrane before passage through a Ni-NTA Sepharose column (GE, Boston, MA, USA). The column was washed with washing buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 20 mmol/L imidazole), and the recombinant alginate lyase AlgB was eluted with elution buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 0.3 mol/L imidazole). The eluate was dialyzed in 0.05 mol/L Tris-HCl buffer (pH 7.5) overnight and analyzed by 12% SDS-PAGE. The protein concentration was measured by the Bradford method.
5
Synthesis and Purification of Alginate Lyase AlyRm3
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The gene AlyRm3 (GenBank accession. CP001807) was synthesized by GENEWIZ (Suzhou, China), based on the predicted sequence of the alginate lyase gene from Rhodothermus marinus DSM 4252. This gene was ligated into the pET-21a (+) vector. The synthetic plasmid was obtained and transformed into receptor cells E. coli BL21 (DE3). The recombinant strain was grown in Luria-Bertani (LB) including 100 μg/mL ampicillin and was incubated at 37 °C for 10–12 h, and it induced expression of the AlyRm3 at 22 °C for 36–48 h after the addition of 0.1 mM IPTG. After protein expression was completed, the AlyRm3 expressed by the bacterium was collected and the crude enzyme solution obtained was purified by Ni-NTA sepharose column (GE Healthcare, Uppsala, Sweden) and Amicon Ultra-15mL, 50 kDa Centrifugal Filter Unit (Millipore, Shanghai, China) [24 (link)]. SDS-PAGE was applied to analyze the purity of the recombinant AlyRm3. Furthermore, the Coomassie brilliant blue G-250 (Beyotime Institute of Biotechnology, Nantong, China) was used to test the concentration of proteins.
6
Recombinant Expression and Purification of Human SIRT6
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The human full-length SIRT6 gene was inserted into the prokaryotic expression vector (pET-28a (+)). The verified recombinant plasmid was transformed into Escherichia coli BL21 (DE3) expression strain and cultured overnight at 37 °C on LB plates supplemented with 50 μg/mL kanamycin. Single colonies were inoculated in fluid kanamycin-containing LB mediums. It was shaken at 160 rpm for 16 h at 37 °C and transferred to kanamycin-containing LB medium at a ratio of 1:100. It was shaken at 160 rpm at 37 °C until the OD600 reached 0.6. The Isopropyl-β-d-thiogalactopyranoside was added to make the final concentration of solution 0.5 mmol/L, cultured for 15 °C and 160 rpm for 24 h.
Bacterial liquid was harvested at 12,000 rpm at 4 °C for 20 min, and then re-suspended in Lysis buffer (20 mmol/L Hepes pH 7.5, 20 mmol/L imidazole, 5% Glycerol, 500 mmol/L NaCl). Cell pellets were disrupted by high-pressure crusher (JNBIO, Guangzhou China) and centrifuged (4 °C, 12000 rpm, for 20 min). Supernatant was collected and purified by FPLC with a 1 mL Ni–NTA Sepharose column (GE Healthcare, Stamford, USA). The SIRT6 protein was eluted at containing 50 mM, 100 mM, 150 mM, 200 mM, and 500 mM imidazole in elution buffer. The purified SIRT6 protein was dialyzed in dialysis buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2). The purified SIRT6 protein was analyzed by SDS-PAGE.
Bacterial liquid was harvested at 12,000 rpm at 4 °C for 20 min, and then re-suspended in Lysis buffer (20 mmol/L Hepes pH 7.5, 20 mmol/L imidazole, 5% Glycerol, 500 mmol/L NaCl). Cell pellets were disrupted by high-pressure crusher (JNBIO, Guangzhou China) and centrifuged (4 °C, 12000 rpm, for 20 min). Supernatant was collected and purified by FPLC with a 1 mL Ni–NTA Sepharose column (GE Healthcare, Stamford, USA). The SIRT6 protein was eluted at containing 50 mM, 100 mM, 150 mM, 200 mM, and 500 mM imidazole in elution buffer. The purified SIRT6 protein was dialyzed in dialysis buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2). The purified SIRT6 protein was analyzed by SDS-PAGE.
7
Recombinant SPANXN4 Protein Purification
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The BL21 (DE3) competent cell line was transformed with the human SPANXN4 gene in the pExp-GST plasmid. Overnight cultures from a single colony were prepared in LB media (Sigma) with ampicillin and incubated at 37°C. The pre-inoculum was inoculated into 4,000 mL LB media with ampicillin. The culture was grown at 37°C to OD600 0.6–0.8, induced with 0.5 mM IPTG, and incubated at 25°C for ∼20 h. Cells were harvested by centrifugation and resuspended in lysis buffer (phosphate buffer pH 7.4 with 300 mM NaCl, lysozyme 0.1 mg/mL, DNase I 0.01 mg/mL, 0.02 mM MgCl2, and protease inhibitor cocktail 1×). The cells were lysed by sonication (5 s ON and 10 s OFF) for 5 min, 40% amplitude. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was purified using an Ni–NTA Sepharose column (GE). The supernatant was passed twice through the column after equilibration with wash buffer (PBS, pH 7.4, containing 10 mM imidazole). The column was washed with 50 column volumes of wash buffer, followed by elution with elution buffer (PBS, pH 7.4 with 500 mM imidazole). The fractions containing SPANXN4 protein were pooled, dialyzed against PBS, and subjected to gel filtration using the HiLoad 26/600 Superdex 200 pg FPLC column. The fractions containing the protein were pooled and quantified using a BCA assay.
8
Purification of Recombinant Hybrid Peptide
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Recombinant hybrid peptide was purified by Ni-NTA Sepharose column (GE Healthcare, Chicago, IL, USA). The expressed culture medium centrifuged at 10,000× g for 15 min, 4 °C, and the supernatant was collected and then filtrated by 0.22 μm filter (Millipore, Burlington, MA, USA). Ni-NTA Sepharose column was pre-equilibrated with buffer containing 20 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, pH 7.5. The supernatants were loaded onto Ni-NTA column and washed with equilibration buffer. The bound peptide then eluted by buffer (20 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, pH 7.5) five times. Fractions were pooled and then concentrated by Amicon Ultra centrifugal filters (Millipore, Burlington, MA, USA). The collected peptide was further purified by reversed-phase high performance liquid chromatography (RP-HPLC). Purified peptide was quantified through Bradford assay kit (Sangon Biotech, Shanghai, China) using bovine serum albumin as a standard and analyzed by 16%/6 M urea Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) [37 (link)].
9
Optimized Alginate Lyase Production
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After codon optimization, the ALYW201 gene with the XPR2 signal peptide gene was synthesized (Synbio Technologies, Suzhou, China). The optimized sequence was with an average GC content of 50% codon and an adaptable Index (CAI) of 0.84, without medium or large hairpins. The synthesized DNA fragment was transformed into the URA-strain [30 (link)]. After an 84 h cultivation in GPPB liquid medium at 30 °C, the alginate lyase activities of the positive transformants were detected. The recombinant strain Y23 showed the highest extracellular activity. During the fermentation period of Y23 at flask, the alginate lyase activity and the biomass were detected every 12 h. All of the data were collected in triplicate.
The supernatant of strain Y23 was adjusted to pH 7.5 and then applied to a Ni-NTA sepharose column (GE Healthcare, Chicago, IL, USA). The Alyw201 enzyme was attached to the gel and then washed off with imidazole solution. The purity and Mw of Alyw201 were verified by SDS-PAGE on 12% (w/v) gel. The catalyzing activities of Alyw201 were performed by using polyM, polyG, and alginate as substrates, respectively, to investigate the substrate preference.
The supernatant of strain Y23 was adjusted to pH 7.5 and then applied to a Ni-NTA sepharose column (GE Healthcare, Chicago, IL, USA). The Alyw201 enzyme was attached to the gel and then washed off with imidazole solution. The purity and Mw of Alyw201 were verified by SDS-PAGE on 12% (w/v) gel. The catalyzing activities of Alyw201 were performed by using polyM, polyG, and alginate as substrates, respectively, to investigate the substrate preference.
10
Purification and Characterization of VaAly2
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After transformation of the recombinant plasmids into E. coli BL21(DE3), the cells were cultivated in LB medium with addition of 50 μg/mL kanamycin at 37°C and 200 rpm before the optical density at 600 nm reached 0.6–0.8. After 20 h of cultivation at 16°C by induction at 0.1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG), the cells were centrifugated and the pellets were resuspended in the lysis buffer [50 mM Tris–HCl, 300 mM NaCl (pH 9.0)] and were further disrupted by an ultrasonic homogenizer. After centrifugation at 10,000 × g for 10 min, the crude enzyme solution was purified by loading onto a Ni-NTA Sepharose column (GE Healthcare, USA). The eluent was desalted with a PD-10 column to eliminate imidazole. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to examine the purified enzymes, and a NanoPhotometer N60 (Implen, Germany) was used to quantify the enzyme concentrations. Furthermore, the oligomeric state of VaAly2 was examined by the size-exclusion chromatography using a HiLoad 16/600 Superdex 200 prep-grade column (GE Healthcare, USA) in 50 mM Tris–HCl (400 mM NaCl, pH 8.0) with a flow rate of 1 mL/min. Myoglobulin (17 kDa), ovalbumin (44 kDa), human albumin (66 kDa) and IgG (158 kDa) from GE Healthcare were used as protein size standards.
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