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

Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion... 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 (IC50) and limit of detection (LOD) for GCA were 216 ng mL1 and 37.0 ng mL1, respectively, and the linear response range extended from 71.0 to 657 ng mL1. 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Analytical Methods Royal Society of Chemistry

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

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Publisher
The Royal Society of Chemistry
Copyright
This journal is © The Royal Society of Chemistry
ISSN
1759-9660
eISSN
1759-9679
D.O.I.
10.1039/c8ay00848e
Publisher site
See Article on Publisher Site

Abstract

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 (IC50) and limit of detection (LOD) for GCA were 216 ng mL1 and 37.0 ng mL1, respectively, and the linear response range extended from 71.0 to 657 ng mL1. 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.

Journal

Analytical MethodsRoyal Society of Chemistry

Published: May 29, 2018

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