Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion protein for glycocholic acid detection in a one-step enzyme-linked immunosorbent assay

• Published in: Analytical methods 2018-01, Vol.1 (22), p.2629-2635
• Main Authors: Cui, Xiping, He, Qiyi, Shen, Ding, Jiang, Zhengyun, Chen, Yingshan, Zhao, Suqing, Hammock, Bruce D
• Format: Article
• Language: English
• Subjects: Acids; Alkaline phosphatase; Binding; Colorimetry; Cross-reactivity; E coli; Electrophoresis; Enzymatic activity; Enzyme activity; Enzyme-linked immunosorbent assay; Enzymes; Fusion protein; Gel electrophoresis; Gene expression; Molecular chains; Molecular weight; Phosphatase; Polymerase chain reaction; Proteins; Sodium; Sodium dodecyl sulfate; Sodium lauryl sulfate; Splicing
• ISSN: 1759-9660; 1759-9679
• DOI: 10.1039/c8ay00848e

A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for glycocholic acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli ( E. coli ). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC 50 ) and limit of detection (LOD) for GCA were 216 ng mL −1 and 37.0 ng mL −1 , respectively, and the linear response range extended from 71.0 to 657 ng mL −1 . The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA. One-step enzyme-linked immunosorbent assay for glycocholic acid based on single-chain variable fragment-alkaline phosphatase fusion protein.