title: High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.
abstract: 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.
authors: Research Institute, Gekkeikan Sake Co. Ltd., 101 Shimotoba-koyanagi-cho, Fushimi-ku, Kyoto, Kyoto 612-8385, Japan. hisada@gekkeikan.co.jp
authors: Hiromoto
authors: H
authors: Hisada
authors: Hiroko
authors: H
authors: Tsutsumi
authors: Hiroki
authors: H
authors: Ishida
authors: Yoji
authors: Y
authors: Hata
mesh_terms: D000818:Animals; D001236:Aspergillus oryzae / Q000235:genetics / Q000378:metabolism*; D002161:Camelids, New World / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins