A construct encoding human complement split product C4dg fused to an N-terminal human CD5 signal peptide for secretion and a C-terminal 6xHIS Tag was cloned into the mammalian expression vector pCEP4 (Thermo Fisher Scientific, Waltham, MA). Transient expression was performed under serum-free conditions in suspension HEK293-6E as described by Margreitter et al. [30 ]. Briefly, transfection of the pCEP4 vector was performed with polyethylemine (PEI; linear 25 kDA, Polysciences) at a cell density of 1x106/ml. At Day 7, supernatants were filtered through a 0.45 μm syringe filter and dialyzed extensively against 50 mM Na-Phosphate pH8.0, 150 mM NaCl prior to HisTALON IMAC purification (see above).
Pcep4
Manufactured by Polysciences
Sourced in United States
The PCEP4 is a laboratory device used for the purification and concentration of proteins, nucleic acids, and other biomolecules. It utilizes a centrifugal force-based filtration process to separate target molecules from unwanted components in a sample.
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2 protocols using pcep4
1
Secreted Human Complement C4dg Construct
2
Cloning and Expression of scFv-Fc Fusion Proteins
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An expression vector modified from pCEP4 (Invitrogen) was used for cloning and has been previously described.14 (link), 15 (link) The vector carries an expression cassette composed of the leader sequence of the human Ig κ-chain, two SfiI sites for insertion of the antibody gene of interest, the hinge region of human IgG1 and the CH2–CH3 domains of rabbit IgG.
The phagemid DNA of selected clones and the expression vectors were digested with SfiI, and the scFv genes were cloned into the expression vector. Recombinant pCEP4 was transfected into HEK 293F cells using 25-kDa linear polyethylenimine (Polysciences, Warrington, PA, USA) as previously described.16 (link) Overexpressed scFv-Fc fusion proteins were purified by affinity chromatography using protein A Sepharose columns (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.
The phagemid DNA of selected clones and the expression vectors were digested with SfiI, and the scFv genes were cloned into the expression vector. Recombinant pCEP4 was transfected into HEK 293F cells using 25-kDa linear polyethylenimine (Polysciences, Warrington, PA, USA) as previously described.16 (link) Overexpressed scFv-Fc fusion proteins were purified by affinity chromatography using protein A Sepharose columns (Repligen, Waltham, MA, USA) according to the manufacturer's instructions.
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