Optimal production of a fusion protein consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in fed-batch culture of Pichia pastoris.
Anticancer Res
; 34(8): 3925-35, 2014 Aug.
Article
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| MEDLINE
| ID: mdl-25075014
ABSTRACT
BACKGROUND/AIM:
The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter. MATERIALS ANDMETHODS:
We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZα-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H.RESULTS:
The highest concentration of secreted fusion protein, 738 ± 44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1.CONCLUSIONS:
The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins.Palabras clave
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Base de datos:
MEDLINE
Asunto principal:
Pichia
/
Proteínas Recombinantes de Fusión
/
Moléculas de Adhesión Celular
/
Interleucina-2
/
Anticuerpos de Cadena Única
/
Antígenos de Neoplasias
Tipo de estudio:
Risk_factors_studies
Límite:
Animals
Idioma:
En
Revista:
Anticancer Res
Año:
2014
Tipo del documento:
Article
País de afiliación:
Tailandia