The recombinant sdAb clones were transformed into E. coli Tuner (DE3) host and plated onto LB agar plates supplemented with Ampicillin and incubated at 37 °C overnight. The next day, a single colony was inoculated into 50 mL Terrific Broth (TB) and shook overnight at 25 °C. The culture was then transferred to a 500 mL TB culture to grow for 3 hours at RT before adding 0.5 mM IPTG to induce protein expression. After 2.5 hrs of IPTG induction, bacterial cells were pelleted down by centrifugation and subjected to osmotic shock and IMAC extraction according to a previously published protocol (Hayhurst et al., 2003 (link); Turner et al., 2014 (link)). Following IMAC extraction, recombinant proteins were further purified from other protein contaminates through Superdex 75 10/300 GL columns (GE Healthcare) operated under a BioLogic DuoFlow chromatography system (Bio-Rad). Protein concentrations were estimated by measuring their absorbance at 280 nm.
Biologic duoflow chromatography system
Manufactured by Bio-Rad
Sourced in United States
The BioLogic DuoFlow chromatography system is a laboratory equipment used for separating and purifying different molecules, such as proteins, nucleic acids, and other biomolecules. It is designed to perform various chromatographic techniques, including ion exchange, size exclusion, and affinity chromatography. The system provides precise control over flow rate, pressure, and other parameters to ensure accurate and reproducible results.
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Lab products found in correlation
Ni nta column, by Qiagen (4 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (3 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
P azido l phenylalanine azf, by Chem-Impex (2 mentions)
Pqe80 plasmid, by Qiagen (2 mentions)
Uno q1 anion exchange column, by Bio-Rad (2 mentions)
Ni nta agarose, by Qiagen (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Vivaspin centrifugal concentrator, by Sartorius (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ni nta column, by GE Healthcare (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Superdex 200 10 300 gl size exclusion column, by GE Healthcare (1 mentions)
Ziptip with c18 resin, by Merck Group (1 mentions)
Hitrap sp hp cation exchange column, by GE Healthcare (1 mentions)
Histrap hp 1 ml column, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
23 protocols using biologic duoflow chromatography system
1
Recombinant sdAb Protein Purification
2
Purification of His-Tagged Proteins
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Plasmids for protein expression were transformed into E. coli BL21 (DE3). Cells containing plasmid were cultivated in 2-L flasks containing 1 L LB medium at 37 °C with 50 mg L−1 kanamycin. When the OD600 reached 0.6–0.8, 0.1 mM IPTG was added to the cultures, which were then grown for an additional 24 h at 16 °C. The cells were harvested and then resuspended in 40 mL buffer A [50 mM Tris-Cl (pH 7.6), 300 mM NaCl, 4 mM β-mercaptoethanol].
The cell suspensions were lysed using a high-pressure homogenizer and centrifuged at 30,000g for 45 min. The supernatant was then filtered, and 6% buffer B (500 mM imidazole in buffer A) was added to adjust the supernatant to 30 mM imidazole to reduce nonspecific binding. Biologic DuoFlow Chromatography System (Bio-Rad) was used for protein purification. The His-tagged protein was purified using a Ni-NTA column (GE Healthcare Life Science, USA) according to the methods described by Zhu10 (link). The protein was then concentrated and preserved in PB buffer (100 mM phosphate buffer with 10% glycerol) for in vitro assays to test the catalysis for different substrates. For kinetic assays, the protein was preserved in 50 mM Tris buffer (50 mM Tris, 10% glycerol). The protein concentration was measured using a Pierce1 BCA protein assay kit (Thermo Fisher Scientific) and recorded on an Enspire Multimode Plate Reader (PerkinElmer, MA, USA).
The cell suspensions were lysed using a high-pressure homogenizer and centrifuged at 30,000g for 45 min. The supernatant was then filtered, and 6% buffer B (500 mM imidazole in buffer A) was added to adjust the supernatant to 30 mM imidazole to reduce nonspecific binding. Biologic DuoFlow Chromatography System (Bio-Rad) was used for protein purification. The His-tagged protein was purified using a Ni-NTA column (GE Healthcare Life Science, USA) according to the methods described by Zhu10 (link). The protein was then concentrated and preserved in PB buffer (100 mM phosphate buffer with 10% glycerol) for in vitro assays to test the catalysis for different substrates. For kinetic assays, the protein was preserved in 50 mM Tris buffer (50 mM Tris, 10% glycerol). The protein concentration was measured using a Pierce1 BCA protein assay kit (Thermo Fisher Scientific) and recorded on an Enspire Multimode Plate Reader (PerkinElmer, MA, USA).
3
Site-Specific Protein Labeling Protocol
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DBCO-PEG4-carboxyrhodamine 110, DBCO-amine, TCO-NHS ester, and tetrazine-DBCO were purchased from Bioconjugate Technology Company (Scottsdale, AZ). p-Azido-L-phenylalanine (AZF) was purchased from Chem-Impex International (Wood Dale, IL) and dissolved in 0.2 M sodium hydroxide to prepare 100 mM stock solution. Ni-NTA agarose and pQE80 plasmid were obtained from Qiagen (Valencia, CA). Vivaspin centrifugal concentrators with a MWCO of 50 kDa were obtained from Sartorius Corporation (Bohemia, NY). ZipTip C18 was purchased from Millipore Corporation (Billerica, MA). Sequencing-grade modified trypsin was obtained from Promega Corporation (Madison, WI). PD-10 desalting columns and Superdex 200 10/300 GL sized exclusion column were obtained from GE Healthcare (Piscataway, NJ). UNO Q1 anion exchange column and Biologic DuoFlow chromatography system were obtained from Bio-Rad (Hercules, CA). All chemicals were obtained from Sigma-Aldrich Corporation (St. Louis, MO) unless otherwise stated.
4
Bioconjugation of Proteins with AzF
5
Purification of Recombinant Protein Bo(Man26B)
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BoMan26B was expressed as described previously (11 (link)) and purified with slight modifications: Escherichia coli BL21(DE3) cells containing expressed protein were dissolved in lysis buffer (20 mm NaH2PO4, 0.5 m NaCl, 1 mm CaCl2, and 20 mm imidazole, pH 7.4) and lysed by a French press. After subsequent centrifugation (JA 25.50 rotor, 21,500 rpm, 4 °C, 30 min), a HisTrapTM HP 1-ml column (GE Healthcare, Pollards Wood, UK) was equilibrated with lysis buffer on a BioLogic DuoFlow chromatography system (Bio-Rad) at 10 °C. The sample was loaded on the column that was washed with 20 ml of lysis buffer, followed by a gradient of 0–100% elution buffer (lysis buffer, with 400 mm imidazole) over 20 ml, collecting 1-ml fractions at 1 ml/min. The purity of the fractions was assessed with SDS-PAGE (Fig. S6 ), and relevant fractions were pooled, and the buffer was changed to 50 mm MES buffer, pH 6.5, with 1 mm CaCl2 (storage buffer) (11 (link)).
6
Purification of Luciferase Enzymes
7
Expression and Purification of OmpA Proteins
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The cloning and expression of full-length OmpA (OmpA325) and the N-terminal barrel domain (OmpA171) have been described previously [7 (link)]. In brief, the gene sequences were PCR-amplified and ligated into a pET11a vector, and the proteins were expressed to inclusion bodies in hms174(DE3) cells with induction by 1 mM IPTG (Fisher). The proteins were purified in Urea Buffer (8 M urea, 20 mM Tris, pH 8) prepared with ultra-pure, deionized urea (Amresco), using anion exchange and gel filtration on a BioRad BioLogic Duoflow Chromatography System as described [7 (link)]. For OmpA325, Urea Buffer contained 2 mM TCEP (Pierce). Protein concentrations were determined by spectroscopy using previously calculated extinction coefficients [7 (link)]. Purified protein was stored in aliquots at -80°C until use.
8
Protein Isolation, Purification, and Characterization
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This research contained five stages which were protein isolation, purification, recovery, characterization, and qualitative analysis. The protein isolation was done by dialysis the sample in 2 L of 50 mM Tris-HCl buffer solution (pH 8.0) to remove honey sugars. The protein purification was done using biologic duoflow chromatography system (Bio-Rad, USA) and an anion exchanger column to bind the targeted protein. The honey proteins were recovered using amicon® ultra centrifugal filters (Merck, Ireland) in order to get bands with higher intensity. The protein characterization was done using SDS-PAGE method to characterize different proteins based on their molecular weight and also to discover which bands had the highest intensity. The highest intensity bands were qualitatively identified by LC-MS/MS instrument. The more detailed process would be explained in the next research expansion section.
9
Recombinant single-domain antibody expression
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Representative sdAb genes from each sequence family were subcloned into a periplasmic expression vector, pET22b, for protein preparation and transformed into Turner (DE3) E. coli strain (EMD Millipore, Billerica, MA). The expression and purification procedure was performed according to the previously published protocol [48 (link), 49 (link)]. Following IMAC extraction, sdAbs were further purified from other protein contaminates or aggregates through gel filtration chromatography using a Superdex 75 10/300 GL column (GE Healthcare) on a BioLogic DuoFLow chromatography system (Bio-Rad). Monomeric sdAb concentration was determined using BCA protein kit (Pierce) or a NanoDrop 1000 instrument.
10
Refolding and Purification of Denatured Proteins
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