Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system.
Protein Expr Purif
; 31(1): 34-41, 2003 Sep.
Article
en En
| MEDLINE
| ID: mdl-12963338
ABSTRACT
We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Interleucina-3
/
Vectores Genéticos
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2003
Tipo del documento:
Article
País de afiliación:
Alemania