High-level expression, purification and study of bioactivity of fusion protein M-IL-2((88)Arg, (125)Ala) in Pichia pastoris.
Protein Expr Purif
; 101: 99-105, 2014 Sep.
Article
en En
| MEDLINE
| ID: mdl-24955549
ABSTRACT
M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.
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Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Neoplasias Ováricas
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Pichia
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Proteínas Recombinantes de Fusión
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Interleucina-2
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Meliteno
Límite:
Female
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Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2014
Tipo del documento:
Article