Expression, purification, and characterization of recombinant human interleukin-13 from NS-O cells.
Interleukin-13 is a cytokine which is secreted by activated T lymphocytes and primarily impacts monocytes, macrophages, and B cells. A synthetic gene coding for human interleukin-13 has been prepared and cloned into expression vector pEE12. The construct was transfected into NS-O cells, which showed stable expression of the recombinant protein. A four-step purification procedure consisting of S-Sepharose, Q-Sepharose, hydroxyapatite, and Sephacryl-100 chromatographies yielded bioactive interleukin-13 of > 98% purity. The purified protein was structurally characterized. The extinction coefficient at 280 nm was determined to be 5678 M-1 cm-1. Amino acid sequencing confirmed that the N-terminus of the purified protein was intact. Electrospray mass spectrometric analysis, size-exclusion chromatography, and SDS-PAGE revealed that the biologically active protein is monomeric and unglycosylated. Mass spectrometry and a chemical assay for free sulfhydryls indicated that the four cysteine residues of interleukin-13 are involved in two intramolecular disulfide bonds. The circular dichroism spectrum confirms that interleukin-13 belongs to the alpha-helical family of cytokines. A biologically inactive covalent trimer also forms in the cell culture, but can be separated from the monomer by the hydroxyapatite and size-exclusion chromatographies. These data indicate that human interleukin-13 retains many structural similarities to human interleukin-4, from which it arose by a gene duplication event.[1]References
- Expression, purification, and characterization of recombinant human interleukin-13 from NS-O cells. Cannon-Carlson, S., Varnerin, J., Tsarbopoulos, A., Jenh, C.H., Cox, M.A., Chou, C.C., Connelly, N., Zavodny, P., Tang, J.C. Protein Expr. Purif. (1998) [Pubmed]
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