Technology of Compact MAb and its Application for Medicinal Plant Breeding Named as Missile Type Molecular Breeding

Waraporn      Putalun

Abstract

Single-chain variable fragment (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the scFv against solamargine (As-scFv) gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 μg/ml. The expressed antisolamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology which can produce a high yield of secondary metabolites.

Keywords: Hairy root cultures, plant breeding, single-chain variable fragment, solasodine glycosides, Solanum khasianum, scFv against solamargine (As-scFv) gene, monoclonal antibody, secondary metabolites, solamargine, chelate affinity column