The expression of pZIP4-ECD was induced by 0.2 mM IPTG at OD600∼0.6 in the strain of Origami B(DE3) pLysS (Novagen) in lysogeny broth medium. pZIP4-ECD was purified using nickel-nitrilotriacetic acid (Ni-NTA) column. After removing the N-terminal His6-tag by thrombin, the protein was further purified by ion exchange chromatography on a Mono-Q column and then by size exclusion chromatography (GE Healthcare). The purified protein was concentrated to 10 mg ml−1 and crystallized by using the sitting-drop method. Leaf-shaped crystals showed up within 2 days in 100 mM MES, 100 mM NH4Cl, 17% PGE 3350, pH 6.5 at 21 °C and grew to full size in 1 week. The crystals were cryo-protected by soaking in 30% of PEG 3350 for a few minutes and flash-frozen in liquid nitrogen. The preparation and crystallization of selenomethionine-substituted protein were the same as the native protein. Expression and purification of pZIP14-ECD was the same as pZIP4-ECD.
Size exclusion chromatography
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Size exclusion chromatography is a technique used to separate molecules based on their size and shape. It allows the separation and purification of macromolecules, such as proteins, nucleic acids, and polymers, from smaller molecules or ions present in a sample. The process involves passing the sample through a porous gel matrix, where larger molecules elute faster than smaller ones, enabling their separation and isolation.
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Lab products found in correlation
Q sepharose, by GE Healthcare (2 mentions)
Mono q column, by GE Healthcare (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Ni nta affinity column, by Qiagen (1 mentions)
Bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
Ni sepharose affinity chromatography, by GE Healthcare (1 mentions)
Quikchange, by Agilent Technologies (1 mentions)
Anion exchange chromatography, by GE Healthcare (1 mentions)
Ni agrose affinity resin, by Qiagen (1 mentions)
Hek293f, by Thermo Fisher Scientific (1 mentions)
Source 15q, by GE Healthcare (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Herceptin, by Roche (1 mentions)
Protein g sepharose, by Roche (1 mentions)
Ni affinity chromatography, by GE Healthcare (1 mentions)
5 ml ni nta column, by GE Healthcare (1 mentions)
Bl21 de3 cells, by Merck Group (1 mentions)
Ni nta column, by GE Healthcare (1 mentions)
Ni nta column chromatography, by GE Healthcare (1 mentions)
Cu400 em grids, by Electron Microscopy Sciences (1 mentions)
20 protocols using size exclusion chromatography
1
Purification and Crystallization of pZIP Protein
2
CFXTEN Fusion Protein Purification
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Example 29
Two CFXTEN constructs with C-terminal XTEN were utilized to establish a purification method. For both pBC0145 with a C-terminal XTEN of 288 amino acids of the AE family (see sequence in Table 21) and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG family (see sequence in Table 21), a tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media from cell culture. Products were then captured using a strong anion exchange chromatography resin, and then further purified using VIIISelect affinity chromatography (GE Healthcare). An additional size exclusion chromatography (GE Healthcare) was applied to FVIII-pBC0146 as a third polish step to remove high molecule weight species. The purity of both fusion proteins was deemed acceptable by HPLC-SEC and was further confirmed by SDS-PAGE analysis of the two CFXTEN constructs showing CFXTEN products at expected sizes. The specific activity of both molecules was comparable to B-domain deleted FVIII, as measured by aPTT coagulation assay and ELISA determination of FVIII concentration.
3
Nanobody Expression and Purification
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Nanobodies were expressed and purified as reported previously31 (link)–33 (link),63 (link). Briefly, bacteria were grown in terrific broth at 37 °C overnight. The following day, a 1:100 culture was grown until an OD of 0.7–0.9 and then induced with 1 mM IPTG. After 20–24 h of shaking at 25 °C, E. coli were pelleted and resuspended in SET buffer (200 mM Tris, pH 8.0, 500 mM sucrose, 0.5 mM EDTA, 1X cOmplete protease inhibitor (Sigma)) for 30 min at room temperature. Following equilibration, bacteria were osmotically lysed with the addition of 2x volume of deionized water rocked for 45 min. Prior to centrifugation at 17,000×g for 20 min, NaCl was added to 150 mM, MgCl2 to 2 mM, and imidazole to 20 mM. The periplasmic fraction was filtered with a 0.22 μm filter and incubated with 4 mL 50% Ni-NTA resin equilibrated in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 40 mM imidazole) (Qiagen) per liter of initial bacterial culture. Supernatant and resin were incubated, rocking for an hour, and then pelleted at 50×g for 1 min. Ni-NTA resin was washed with 10 volumes of wash buffer before elution (20 mM HEPES, pH 7.5, 150 mM NaCl, 250 mM imidazole). Eluted protein was concentrated with 3 kDa MWCO filters (Amicon) before size-exclusion chromatography (GE Healthcare). Proteins were stable at 4 °C.
4
Structural Analysis of LasR Ligand Complexes
5
Recombinant fHBP Variant Purification and Characterization
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Nonlipidated recombinant fHBP (rP2086) variants were expressed and purified as previously described (25 (link)). Mutations in the MN86-994-11 binding epitope were introduced by site-directed mutagenesis. In this case, a His-tagged version of rP2086-B01 cloned into plasmid vector pET30a was used as the mutagenesis template to facilitate the purification of recombinant mutants. A mutagenesis kit was used in accordance with the manufacturer’s instructions (QuikChange; Agilent), mutagenic oligonucleotides used in the reaction were designed with the QuikChange Primer Design Program, and the presence of intended mutations and the absence of secondary mutations were confirmed by DNA sequencing. Mutant proteins expressed in Escherichia coli BL21(DE3) were purified by Ni Sepharose affinity chromatography and size exclusion chromatography (GE Healthcare). All CD and ITC experiments were done with 1× PBS, pH 7.4. Protein and antibody samples were thoroughly dialyzed against experimental buffer. Concentrations of rP2086-B01 and MN86-994-11 were determined spectrophotometrically by using extinction coefficients of 0.363 and 1.4 (mg/ml)−1 cm−1 at 280 nm, respectively. Light scattering was taken into account as previously described (42 (link)).
6
Recombinant HLA Protein Production
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Briefly, plasmids encoding human β2m and the extracellular portion of α chain of defined HLA molecules were transformed into Escherichia coli BL21 for expression of the HLA subunits26 (link). Protein inclusion bodies were extracted and refolding of peptide-HLA complexes performed in vitro by rapid dilution. Concentrated, dialyzed HLA proteins were biotinylated in vitro by recombinant biotin ligase (Avidity). Purification was performed by Fast Protein Liquid Chromatography followed by size exclusion chromatography (GE Healthcare Life Sciences).
7
Purification of Human MTH1 Protein
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The gene of the human mutT homologue MTH1 was ligated into PET 28a vector (Novagen). After the recombinant plasmid was verified by sequencing, it was transformed into E. coli strain BL21 Star (Invitrogen) at 293K, which were grown in LB medium at 37°C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) at 18°C for 16 hours. Bacterial cells were lysed by ultrasonification on ice in buffer containing 100 mM Tris-HCl pH 8.8, 200 mM NaCl, 10% glycerol, 1% TritonX100, 5 mM β-mercaptoethanol. Soluble N-terminally hexa-histidine tagged MTH1 was bound to Ni-agrose affinity resin (Qiagen), washed with a buffer containing 20 mM Tris-HCl pH 8.8, 200 mM NaCl and 10 mM imidazole and eluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl, and 150 mM imidazole. The eluted protein was concentrated and diluted with a buffer containing 20 mM Tris-HCl pH 8.8, 250 mM NaCl and digested with thrombin for 12–15 h at 277 K. Cut MTH1 was purified by Ni-agrose affinity resin (Qiagen). The protein was further purified with anion exchange chromatography (GE Health), using a linear gradient of 10 mM to 1 M NaCl concentration and size exclusion chromatography (GE Health) at 20 mM Tris-HCl pH 8.8 and 200 mM NaCl [40 (link)].
8
Recombinant Protein Purification Protocols
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Recombinant proteins were expressed in HEK-293F (Life Technologies) cells following transient transfection with the Gibco Expi293 expression system kit (Life Technologies). The MST-HN mutations reduce binding of the Fc region to protein G-Sepharose and Seldegs were therefore purified using an anion exchange column (SOURCE-15Q, GE Healthcare) at pH 8.0 and a linear salt gradient (0–0.5 M NaCl). HER2-WT and MOG-WT were purified using protein G-Sepharose (GE Healthcare). 8-18C5 was expressed in recombinant form and purified using protein G-Sepharose23 (link), and clinical grade TZB (Herceptin; Roche) was obtained from the UT Southwestern Medical Center Pharmacy. Recombinant Abdeg (MST-HN, hen egg lysozyme-specific) was purified from culture supernatants using lysozyme-Sepharose1 (link). All recombinant proteins were purified using size-exclusion chromatography (GE Healthcare) in PBS (Lonza) before use in experiments.
9
Recombinant Protein Expression and Purification
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Recombinant Tcnα, TcdB, TpeL, TcsL, and TcsH were expressed in Bacillus subtilis SL401 and purified as His-tagged proteins52 (link). In brief, B. subtilis cells were cultured at 37 °C till OD600 reached 0.6 and then induced with 1 mM isopropyl-β-D-thiogalactoside at 25 °C for 20 h. The recombinant LDLRLA-Fc with His-tag at C-terminus was expressed in the Expi293F cells. In brief, 5 × 108 Expi293F cells were transfected with 750 μg of pHLsec-LDLRLA-Fc using 1 mg/ml PEI. The supernatant was collected 4 days post-transfection and applied to purification. All above recombinant proteins were purified by Ni-affinity chromatography and size-exclusion chromatography (GE Healthcare).
10
Overexpression and Purification of Cry5B
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A pET28b plasmid containing the cry5B(1–772) gene was transformed into E. coli BL21(DE3) and the recombinant protein was overexpressed in Luria–Bertani (LB) media supplemented with 50 μg/mL kanamycin and induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 °C and grown overnight. The harvested cells were lysed by sonication in buffer A (20 mM Tris pH 8.0, 500 mM NaCl, 0.1% phenylmethylsulfonyl fluoride (PMSF) and 0.1% benzydamine) and loaded on to a 5 mL Ni-NTA column (GE Healthcare), which was then washed with buffer containing a 0 to 500 mM imidazole gradient. The recombinant Cry5B(1–772) protein was eluted at ~150 mM imidazole and further purified by size-exclusion chromatography (GE Healthcare) with buffer B (20 mM HEPES pH 8.0, 50 mM NaCl). The fractions were analyzed by SDS-PAGE and checked by mass spectrometry-based protein identification.
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