WT human cdc42 (Uniprot ID: P60953) was inserted into the pTYB11 IMPACT bacterial expression vector (New England BioLabs), using the Sal1 and SapI restriction enzyme cleave sites. Cdc42 was transformed into Rosetta (DE3) cells (Novagen) that were subsequently cultured in Terrific Broth medium, supplemented with 100 μg ml−1 carbenicillin at 37 °C. Protein expression was induced with 1 mM IPTG at 18 °C for 16 h. Cells were homogenized in lysis buffer (25 mM Tris pH 8.0, 300 mM NaCl, 5 mM MgCl2, 0.1 mM GDP, 4 mM benzamidine hydrochloride, and 1 mM PMSF) and lysed using a microfluidizer 110L (Microfluidics). Lysates were clarified by centrifugation at 16,000g for 45 min, and the supernatant was loaded onto the chitin affinity resin (New England BioLabs). The affinity tag was removed by inducing self-cleavage of the intein domain with 50 mM DTT for 48 h at 4 °C. Proteins were eluted from the column in 25 mM Tris pH 8.0, 300 mM NaCl, 5 mM MgCl2, 0.1 mM GDP, and 50 mM DTT. A final size-exclusion chromatography purification step was performed on a HiLoad 26/600 Superdex 75 pg column (Cytiva) equilibrated in 25 mM Tris pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.1 mM GDP, and 1 mM DTT. The protein was spin concentrated using an ultracentrifugation filter and flash frozen in liquid nitrogen.
Hiload 26 600 superdex 75 pg column
Manufactured by Cytiva
Sourced in Germany
The HiLoad 26/600 Superdex 75 pg column is a size exclusion chromatography column used for protein purification. The column has a bed volume of 320 mL and is packed with a matrix composed of cross-linked agarose and dextran.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by Cytiva (2 mentions)
Chitin affinity resin, by New England Biolabs (1 mentions)
Monos 10 100 gl column, by Cytiva (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Histrap hp, by Cytiva (1 mentions)
Amicon 100 kda molecular weight cut off device, by Merck Group (1 mentions)
Superose 6 increase 3.2 300 column, by Cytiva (1 mentions)
Hbs ep, by Teknova (1 mentions)
Mabselect sure column, by Cytiva (1 mentions)
5 protocols using hiload 26 600 superdex 75 pg column
1
Cdc42 Bacterial Expression and Purification
2
Purification and Characterization of YwfG Variants
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Total bacterial DNA was isolated by using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Primers were designed using the coding sequence (CDS) of locus tag LLG50_11005 of the G50 genome sequence (accession number CP025500.1) (S1 Table ).
DNA fragments encoding variants of YwfG (residues 28–270, 28–336, 28–511, and 860–1034) were amplified by polymerase chain reaction using the primer sets Fw28–Rv270, Fw28–Rv336, Fw28–Rv511, and Fw860–Rv1034, respectively. The resulting fragments were inserted in NdeI/BamHI-digested pET28b (Merck) by In-Fusion cloning reactions (In-Fusion HD Cloning Kit, Takara Bio, Kusatsu, Japan) with an N-terminal His6-tag. The resulting constructs were transformed into E. coli BL21(DE3). Recombinant YwfG variants were expressed at 16°C overnight in the presence of 0.2 mM isopropyl β-d -thiogalactoside and purified by affinity chromatography using a nickel-chelating column (HisTrap HP, Cytiva), followed by gel filtration using a HiLoad 26/600 Superdex 75 pg column (Cytiva) and cation exchange chromatography using a Mono S 10/100 GL column (Cytiva). The obtained recombinant YwfG variants were named YwfG28–270, YwfG28–336, YwfG28–511 (numbers indicate amino acid residues), and MubR4 (residues 860–1034).
DNA fragments encoding variants of YwfG (residues 28–270, 28–336, 28–511, and 860–1034) were amplified by polymerase chain reaction using the primer sets Fw28–Rv270, Fw28–Rv336, Fw28–Rv511, and Fw860–Rv1034, respectively. The resulting fragments were inserted in NdeI/BamHI-digested pET28b (Merck) by In-Fusion cloning reactions (In-Fusion HD Cloning Kit, Takara Bio, Kusatsu, Japan) with an N-terminal His6-tag. The resulting constructs were transformed into E. coli BL21(DE3). Recombinant YwfG variants were expressed at 16°C overnight in the presence of 0.2 mM isopropyl β-
3
Heterologous Expression and Purification of 4F2hc-LAT2 Complex
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To heterologously overexpress human 4F2hc-LAT2, a previously characterized and reported P. pastoris clone was used12 (link),14 (link),37 (link). The recombinant protein was produced in P. pastoris as described23 (link), solubilized with GDN and purified according to published protocols37 (link),38 (link). The anticalin D11vs was produced at preparative scale via secretion in E. coli KS272 with a C-terminal Strep-tag II using the plasmid pNGAL98-D11vs36 (link). After periplasmic protein extraction, the recombinant protein was purified using a Strep-Tactin Sepharose column (IBA, Göttingen, Germany) and finally subjected to SEC in PBS (4 mM KH2PO4, 160 mM Na2HPO4, 115 mM NaCl pH 7.4) on a HiLoad 26/600 Superdex 75 pg column (Cytiva, Freiburg, Germany). The GDN-solubilized 4F2hc-LAT2 heterodimer was mixed with 4 molar equivalents of D11vs and incubated on ice for 1 h followed by concentration to ~ 10 mg/mL with an Amicon 100-kDa molecular weight cut-off device (Merck, Switzerland). This sample was further purified by SEC using a Superose 6 Increase 3.2/300 column (Cytiva, Switzerland) with 20 mM Bis–Tris propane pH 8.0, 150 mM NaCl, 1% (v/v) glycerol, 0.02% (w/v) GDN as running buffer. Individual fractions were analyzed by SDS-PAGE and used for cryo-EM grid preparation.
4
Expression and Purification of GIPR and GLP-1R Ectodomains
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The isolated ECDs of human GIPR (amino acids 26 to 138) and GLP-1R (amino acids 24 to 145) were expressed as 6His-tagged secreted proteins in CHO cells. DNA encoding each protein was transfected into CHO cells for expression. After expression, the proteins were purified by affinity chromatography (HIS-Trap Ni, Cytiva) followed by size exclusion chromatography using a HiLoad 26/600 Superdex 75pg column (Cytiva) with PBS buffer. Fractions were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and fractions meeting purification criteria were pooled, then 0.22 μm filtered. Surface plasmon resonance measurements were made using a Biacore T200 (Cytiva) and analyzed using T200 Evaluation Software Version 3.1. Receptor ectodomain proteins were covalently immobilized (∼250 resonance units) on Sensor Chip CM4 BR100534 using the Amine coupling Immobilization Wizard in the Biacore T200 Control Software Version 2.0.2. Running buffer was HBS-EP (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% P20 [pH 7.6]; Teknova). Concentration series for each sample were made by serial dilution in running buffer. The samples were injected for 150 sec at a 30-µL/min flow rate, followed by 300 sec of dissociation. The surface was regenerated between samples with 10 mM glycine (pH 1.7).
5
Secretion and Purification of Tri- and Tetravalent Nanobodies
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Tri-and tetravalent nanobody DNA received in the pSS1 vector contains an N-terminal BCL1 signal sequence that targets the nanobody for secretion from the cells, allowing their extraction from the cell culture media. Mammalian HEK293T cells were cultured at 37 °C and 5% CO 2 in complete Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin, and 1% glutamine. Tri-and tetravalent nanobody DNA was transiently transfected at 60% cell confluency using polyethylenimine (PEI Max MW 40 000, Polysciences) according to the manufacturer's instructions. Nanobodies were expressed as secreted protein into the culture media, collected after 7 days, and subsequently purified using affinity chromatography using either nickel NTA beads for the trivalent nanobodies or a MabSelect SuRe column (Cytiva) for the Fc-tetravalent nanobodies. Tag cleavage was achieved as described for the divalent nanobodies [13 13. Maqsood, Z. ]. Further purification by size exclusion chromatography on a HiLoad 26/600 Superdex 75 pg column (Cytiva) was used, if necessary, to obtain pure nanobodies. The concentration of purified nanobody was determined using a NanoDrop spectrophotometer (ND-1000, Geneflow) measuring absorbance at 280 nm according to the manufacturer's protocol. Purified nanobodies were stored at -80 °C.
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