期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 24, 期 3, 页码 283-290出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzq107
关键词
agonist; bacterial expression; CAT selection; IL-15; solubility
资金
- Region Pays de la Loire, France
- Ligue Nationale Contre le Cancer (LNCC)
Directed evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15R alpha Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12- to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15R alpha and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15R alpha and acts as an effective agonist of IL-15.
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