After codons were optimized, the TANRD gene containing the XPR2 signal peptide gene was synthesized by Synbio Technologies, China. The average GC content of the optimized sequence was 50% codon and its adaptable index (CAI) was 0.84. Besides, there were no medium or large hairpins in it. The DNA fragment synthesized in this study was transformed into the URA-strain [30 (link)]. After the cultivation in GPPB liquid medium at 30 °C for 48 h, the positive transformants were detected for their tannase activities. Specifically, the recombinant strain T73 had the highest extracellular activity. During the T73 fermentation at flask, the TanRd activity and biomass were determined every 12 h. All the data were collected in triplicate [30 (link)]. The supernatant of strain T71 was adjusted until pH 7.5 and then loaded on a gel filtration chromatography column as well as an anion exchange chromatography column (GE Healthcare, Chicago, IL, USA). The TanRd can be attached to the gel and was then washed off. The Mw and purity of TanRd were verified based on SDS-PAGE on 12% (w/v) gel.
Gel filtration chromatography column
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A gel filtration chromatography column is a laboratory equipment used for the separation and purification of molecules based on their size and shape. It consists of a cylindrical column packed with a porous gel material that allows smaller molecules to enter the pores and travel more slowly through the column, while larger molecules pass through more quickly. This process allows for the effective separation and isolation of different components within a sample.
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Lab products found in correlation
8 protocols using gel filtration chromatography column
1
Optimized TANRD Gene Expression and Purification
2
Optimized Tannase Enzyme Production
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When the codon has been optimized, the gene synthesis embodying signal peptide was completed by Synbio Technologies, which was further transformed into the uracil defect type strain URA-. After 36-h culturing in GPPB media at 35°C, the positive transformant signal of TanF tannase activity were detected (Zhang et al., 2018 (link)). Additional, after the strain with the optimal extracellular activity was fermented in a flask for 96 h, the supernatant was then subjected to gel filtration chromatography column (GE Healthcare, United States) after adjusting the pH to 5.0. In the following of eluting with imidazole solution, sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to determine the purity and Mw (Molecular weight) of TanF.
3
Liposomal Drug Encapsulation and Quantification
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The non-encapsulated drugs were removed from the liposomes after passage through a gel filtration chromatography column (GE Healthcare, UK), with 5 kDa cut-off (PD-10 Desalting columns containing 8.3 mL of Sephadex™ G-25 Medium) and eluted with PBS buffer for all liposome. The concentration of MTX and TAM encapsulated was measured by proton nuclear magnetic resonance (1H NMR) using a Bruker Avance III Instrument, operating at 400 MHz (Guimarães et al., 2019 (link)). Powder liposomes containing drug were dissolved in deuterium oxide (for MTX) or deuterated chloroform (for TAM) to determine the amount of drug in the liposomal formulation. Pyridine was used as internal standard. Quantification of DOX was evaluated by UV–vis spectrophotometry measuring the absorbance at 490 nm. UV–vis spectra of liposomes encapsulated DOX were recorded on spectrophotometer BioTek Synergy™ HT using a plastic microplate. The final DOX concentration was determined based on the respective calibration curve.
The drug leakage was determined as follows:
The drug leakage was determined as follows:
4
Folate-Targeted Liposomal Methotrexate
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Liposomes composed of DOPE/Cholesterol/DSPE-mPEG (54:36:10, molar ratio) [21 (link)] were produced by a pre-concentration ethanol injection method [24 (link)]. Briefly, the lipid components were dissolved in ethanol (20% of the final volume) to obtain a 1:1 initial ratio of organic:aqueous phase (v/v). The organic phase was added to an aqueous phase containing the drug MTX and folate-peptide (0.75% w/v) dissolved in phosphate-buffered saline (PBS) buffer, under vigorous magnetic stirring, at 70 °C. After ethanol reduction, the liposomal suspension was diluted five times with PBS buffer. The non-encapsulated MTX and residual ethanol were removed from the liposomes after passage through a gel filtration chromatography column (GE Healthcare, Hatfield, UK), with 5 kDa cut-off (PD-10 Desalting Columns containing 8.3 mL of Sephadex™ G-25 Medium). After separation, the concentration of the encapsulated MTX was determined by measuring the absorbance at 303 nm, the maximum wavelength of MTX in PBS. The data were recorded on spectrophotometer BioTek Synergy™ HT (Winooski, VT, USA) using a quartz microplate.
5
Preparation of Targeted Liposomal Formulations
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Liposomes were prepared using a thin film hydration method. The formulations produced are based on DOPE/CH/DSPE-MPEG which may contain FA-peptide for specific targeting and/or FITC/Alexa Fluor 647 for fluorescence labelling and/or MTX as drug (Table I). Briefly, known amounts of DOPE, CH and DSPE-MPEG were dissolved in chloroform in a 50 mL round-bottom flask. For macrophage internalization studies, FITC was incorporated in the lipidic film.
The organic solvent was evaporated using a rotary evaporator followed by additional evaporation under reduced pressure by a high-vacuum system to remove remaining traces of chloroform. The resultant dried lipid film was dispersed in phosphate buffered saline (PBS) buffer containing either peptides, Alexa Fluor 647 or the MTX. The mixture was vortex-mixed at a temperature greater than the phase-transition temperature (room temperature) to yield multilamellar vesicles, which were then extruded (extruder supplied by Lipex Biomembranes Inc., Canada) through 200 nm pore size polycarbonate filters (Nucleopore, USA) followed by several passages through 100 nm polycarbonate filters (Nucleopore, USA) to form large unilamellar vesicles. The free peptide, Alexa Fluor 647 and the MTX that was not incorporated into liposomes was removed from the samples after passage through a gel filtration chromatography column (GE Healthcare, USA).
The organic solvent was evaporated using a rotary evaporator followed by additional evaporation under reduced pressure by a high-vacuum system to remove remaining traces of chloroform. The resultant dried lipid film was dispersed in phosphate buffered saline (PBS) buffer containing either peptides, Alexa Fluor 647 or the MTX. The mixture was vortex-mixed at a temperature greater than the phase-transition temperature (room temperature) to yield multilamellar vesicles, which were then extruded (extruder supplied by Lipex Biomembranes Inc., Canada) through 200 nm pore size polycarbonate filters (Nucleopore, USA) followed by several passages through 100 nm polycarbonate filters (Nucleopore, USA) to form large unilamellar vesicles. The free peptide, Alexa Fluor 647 and the MTX that was not incorporated into liposomes was removed from the samples after passage through a gel filtration chromatography column (GE Healthcare, USA).
6
Liposomal Drug Encapsulation Analysis
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The non-encapsulated drugs were removed from the liposomes after passage through a gel filtration chromatography column (GE Healthcare, UK), with 5 kDa cut-off (PD-10 Desalting Columns containing 8.3 mL of Sephadex™ G-25 Medium). After separation of the free drug from liposomal formulation, the concentration of each drug encapsulated in liposomes was determined using three different techniques: 1 H NMR, UV-vis spectrophotometry and HPLC/UV-vis.
7
Purification of BAZ2A and MBD2 Proteins
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Human BAZ2A (aa 536–653) and MBD2 (aa 143–220) were subcloned into the pET28-MHL vector to generate N-terminal His-tagged fusion proteins with a tobacco etch virus cleavage site. The BAZ2A and MBD2 mutants were obtained by QuikChange site-directed mutagenesis (Agilent Technologies) using the WT BAZ2A (aa 536–653) and MBD2 (aa 143–220) expression constructs as templates, respectively. The plasmids were transformed into Escherichia coli BL21 (DE3), and the cells were induced with 0.5 mM IPTG at 14 °C overnight. The harvested cells were resuspended in the buffer containing 20 mM Tris HCl, pH 7.5, 500 mM NaCl, and 5% glycerol, which was then sonicated to break the cells. The supernatant of the cell lysis was collected after centrifugation at 10,000 rpm and further purified using the Ni-NTA resin (Qiagen). Purified proteins were treated with Tobacco etch virus protease to remove the His-tag followed by affinity chromatography, anion-exchange chromatography, and gel-filtration column chromatography (GE Healthcare). Finally, the purified proteins were concentrated to ∼10 mg/ml in 20 mM Tris HCl, pH 7.5, and 150 mM NaCl, with or without 1 mM DTT.
8
Recombinant Expression and Purification of TFAP2A and TFAP2B
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Human TFAP2A (aa 203–420 and aa 279–411) and TFAP2B (aa 219–457) fragments were cloned into the pET28-MKH8-SUMO vector to generate N-terminal His6 and Sumo-tagged fusion proteins with a tobacco etch virus (TEV) cleavage site. Mutants of TFAP2A and TFAP2B were constructed using site-directed mutagenesis and confirmed by sequencing. All of the recombinant plasmids were overexpressed using E. coli BL21 (DE3) under the induction with 0.5 mM IPTG at 14°C overnight. The cells were harvested and then resuspended in a lysis buffer with 500 mM NaCl, 20 mM Tris (pH 7.5) and 5% glycerol, followed by sonication at 4°C. After centrifugation at 16 000 g, the supernatant was collected and further purified using the Ni-NTA resin (Qiagen). The recombinant protein was eluted and treated with TEV protease to remove the His6 and Sumo-tag followed by affinity chromatography, anion-exchange chromatography, and gel-filtration column chromatography (GE Healthcare). The final purified wild-type and mutant proteins of TFAP2A and TFAP2B were stored in a buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl. For ITC binding assay, TFAP2A and TFAP2B proteins were stored in a buffer containing 20 mM Tris (pH 7.5) and 200 mM NaCl.
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