High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.
Appl Microbiol Biotechnol
; 97(2): 761-6, 2013 Jan.
Article
en En
| MEDLINE
| ID: mdl-22752366
Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Aspergillus oryzae
/
Proteínas Recombinantes de Fusión
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Región Variable de Inmunoglobulina
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Camélidos del Nuevo Mundo
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Cadenas Pesadas de Inmunoglobulina
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Alemania