IL-2/Fc fusion proteins were expressed using the Expi293 expression system according to manufacturer instructions (Thermo Scientific). Proteins were constructed as human IgG1 Fc fusions at the N- or C terminus to human IL-2 through a (G4S)4 linker. C-terminal fusions omitted the C-terminal lysine residue of human IgG1. The AviTag sequence GLNDIFEAQKIEWHE was included on whichever terminus did not contain IL-2. Fc mutations to prevent dimerization were introduced into the Fc sequence (Ishino et al., 2013 (link)). Proteins were purified using MabSelect resin (GE Healthcare). Proteins were biotinylated using BirA enzyme (BPS Biosciences) according to manufacturer instructions, and extensively buffer-exchanged into phosphate buffered saline (PBS) using Amicon 10 kDa spin concentrators (EMD Millipore). The sequence of IL-2Rβ/g Fc heterodimer was based on a reported active heterodimeric molecule (patent application US20150218260A1), with the addition of (G4S)2 linker between the Fc and each receptor ectodomain. The protein was expressed in the Expi293 system and purified on MabSelect resin as above. IL2-Rα ectodomain was produced with C-terminal 6xHis tag and purified on Nickel-NTA spin columns (QIAGEN) according to manufacturer instructions.
Bira enzyme
Manufactured by BPS Biosciences
BirA enzyme is a biotin ligase enzyme that catalyzes the biotinylation of target proteins. It attaches biotin, a small molecule that serves as a label or tag, to specific lysine residues in the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Amicon 10 kda spin concentrators, by Merck Group (3 mentions)
Nickel nta spin columns, by Qiagen (3 mentions)
Mabselect resin, by GE Healthcare (3 mentions)
Mabselect, by Cytiva (3 mentions)
Expi293 expression system, by Thermo Fisher Scientific (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Expifectamine, by Thermo Fisher Scientific (1 mentions)
4 protocols using bira enzyme
1
Expression and Purification of IL-2 Fusion Proteins
2
Dimerized Receptor Constructs for IL-2 Signaling
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DNA sequences encoding all proteins were synthesized at Twist Bioscience in pcDNA3.1 (Thermo Fisher Scientific) and transfected into Expi293 cells using ExpiFectamine (Thermo Fisher Scientific), as described by the manufacturer. Fc–IL-2 fusion protein (G4S)4 linkers, with or without a C-terminal AviTag, were expressed in HEK293T cells and purified using protein A chromatography. To create a CD122/CD132 stable dimer, a knob-hole format was used with the extracellular domain of CD122 (GenBank: KAI2597668.1) fused through a (G4S)2 linker to knob human IgG1 Fc (N297G, S354C, T366W) and the extracellular domain of CD132 (GenBank: NP_000197.1) through a (G4S)2 linker to the hole human IgG1 Fc (N297G, Y349C, T366S, L368A, Y407V). The Fc mutations were made to preference heterodimer formation over the formation of homodimers (so-called “knobs-in-hole”) (89 (link)). Plasmids were cotransfected, and CD122/CD132 dimer was purified from the supernatant using protein A chromatography. The ectodomain of recombinant human CD25 was obtained from R&D Systems. WT Fc–IL-2 differed from mIL-2 solely at amino acid position 16. AviTag Fc–IL-2 fusion proteins were site-specifically biotinylated using the BirA enzyme as described by the manufacturer (BPS Bioscience).
3
Expression and Purification of IL-2 Fusion Proteins
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IL-2/Fc fusion proteins were expressed using the Expi293 expression system according to manufacturer instructions (Thermo Scientific). Proteins were constructed as human IgG1 Fc fusions at the N- or C terminus to human IL-2 through a (G4S)4 linker. C-terminal fusions omitted the C-terminal lysine residue of human IgG1. The AviTag sequence GLNDIFEAQKIEWHE was included on whichever terminus did not contain IL-2. Fc mutations to prevent dimerization were introduced into the Fc sequence (Ishino et al., 2013 (link)). Proteins were purified using MabSelect resin (GE Healthcare). Proteins were biotinylated using BirA enzyme (BPS Biosciences) according to manufacturer instructions, and extensively buffer-exchanged into phosphate buffered saline (PBS) using Amicon 10 kDa spin concentrators (EMD Millipore). The sequence of IL-2Rβ/g Fc heterodimer was based on a reported active heterodimeric molecule (patent application US20150218260A1), with the addition of (G4S)2 linker between the Fc and each receptor ectodomain. The protein was expressed in the Expi293 system and purified on MabSelect resin as above. IL2-Rα ectodomain was produced with C-terminal 6xHis tag and purified on Nickel-NTA spin columns (QIAGEN) according to manufacturer instructions.
4
Purification and Characterization of IL-2/Fc Fusion Proteins
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The Expi293 expression system was used to express IL-2/Fc fusion proteins. Expression was conducted as prescribed by the manufacturer instructions (Thermo Scientific). Proteins were formulated as the Fc of human IgG1 fused at its N-or C-terminus to human IL-2 using a (G4S)4 linker. C-terminal lysine residues of human IgG1 were not included in C-terminal fusions. The AviTag sequence GLNDIFEAQKIEWHE was added to the Fc terminus which did not contain IL-2. Fc mutations which prevented dimerization were introduced into the Fc sequence for monovalent muteins 61 . MabSelect resin (GE Healthcare) was used to purify protein. Biotinylation of proteins was conducted using BirA enzyme (BPS Biosciences) according to manufacturer instructions. Extensive buffer-exchanging into phosphate buffered saline (PBS) was conducted using Amicon 10 kDa spin concentrators (EMD Millipore). The sequence which was used to express the IL2Rβ/γ Fc heterodimer was the same as that of a reported, active heterodimeric molecule (patent application US20150218260A1); a (G4S)2 linker was added between the Fc portion and each receptor ectodomain. The Expi293 system was used to express the protein, which was subsequently purified on MabSelect resin as above. The IL2Rα ectodomain was generated to include a C-terminal 6xHis tag and then purified on Nickel-NTA spin columns (Qiagen) according to manufacturer instructions.
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