Evolution of interleukin-15 for higher E. coli expression and solubility.
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-15Rα 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-15Rα and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Rα and acts as an effective agonist of IL-15.
Faculté des Sciences et des Techniques, Université de Nantes, UMR CNRS 6204, Biotechnologie, Biocatalyse, Biorégulation, 2 Rue de la Houssinière, BP 92208, Nantes F-44322, France.
Ghislaine
G
Béhar
Véronique
V
Solé
Alain
A
Defontaine
Mike
M
Maillasson
Agnès
A
Quéméner
Yannick
Y
Jacques
Charles
C
Tellier
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D019409:Interleukin-15 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D053720:Interleukin-15 Receptor alpha Subunit / Q000378:metabolism; D009154:Mutation; D012995:Solubility; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects
D019409:Interleukin-15; D053720:Interleukin-15 Receptor alpha Subunit; D015500:Chloramphenicol O-Acetyltransferase