SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm.
J Biosci Bioeng
; 126(6): 705-709, 2018 Dec.
Article
em En
| MEDLINE
| ID: mdl-30056072
ABSTRACT
Antibody-enzyme fusion proteins have been used for various immunological detection techniques, such as ELISA, Western blotting and so on. The use of genetically-fused antibody-enzyme complexes has advantages over conventional chemical conjugation methods, as they require no complex chemical reactions and allow for the strict control of the number of enzymes fused with antibodies, resulting in a more stable performance of the bifunctional protein. Here, we describe efficient cytoplasmic soluble expression of an antigen-binding fragment (Fab) fused with Escherichia coli alkaline phosphatase (AP), N-terminal Ser-Lys-Ile-Lys (SKIK) tag that can improve the synthesis of the tagged protein, as well as leucine zipper (LZ) to enhance the association of the light chain and the heavy chain of Fab. Our results demonstrated that the SKIK-Fab-LZ-AP fusion was well expressed in E. coli oxidative cytoplasm in soluble form having both antigen-binding and AP activity, and was purified to homogeneity by two step column chromatography, suggesting that the combination of the SKIK tag and AP fusion can greatly increase the productivity and solubility of the Fab-enzyme fusion in an E. coli cytoplasmic expression system.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Fragmentos Fab das Imunoglobulinas
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Fosfatase Alcalina
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Escherichia coli
Idioma:
En
Revista:
J Biosci Bioeng
Assunto da revista:
ENGENHARIA BIOMEDICA
/
MICROBIOLOGIA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Japão