Constructions and expressions of anti-ABA scFv genes in bacteria, yeast and plants
thesisposted on 15.12.2014, 10:33 by Chon-Seng. Tan
Three anti-ABA single-chain Fv (scFv) antibody genes, namely ABA26, MAC61, MAC252 scFvs, had been constructed from mouse and rat hybridomas and expressed in bacteria, yeast and plants. All of these scFv genes could be expressed in E. coli using the T7 promoter, either targeted to the E. coli periplasm or cytoplasm, albeit at comparatively low levels. The cytoplasmically located scFv proteins were in the form of insoluble fraction and did not therefore exhibit any binding activities to the ABA. The majority of the periplasmically located scFv proteins were retained in the bacterial cytoplasm as insoluble bodies with the ompA signal peptide attached to them. Nevertheless, the positive signal in ELISA test indicated that a small portion of scFv proteins were in the form of soluble functional scFv proteins. All the three periplasmically expressed scFv proteins had specific ABA binding activities. However, the affinity constants of the scFv proteins were found to be 5 to 10 fold lower than those of their parental MAbs. In addition, the MAC61 and MAC252 scFv proteins possessed less specific binding sites compared to their parental monoclonal antibodies. In the yeast expression, although no scFv proteins were detected in the case of ABA26 and MAC252 scFvs with the Pichia pastoris expression system, MAC61.scFv proteins were expressed and some of the product was secreted into the culture media. The expression of ABA26, MAC61, and MAC252 scFv genes in plants, either by transient expression using the PVX viral vector or by stable Agrobacterium -mediated transformation, did not result in the accumulation of scFv protein to a detectable level either targeted in cells cytosol, secreted to the apoplast or retented in the endoplasmic reticulum. The low expression levels in E. coli, the difficulties encountered in the P. pastoris system, and the inability plants to express ABA26, MAC61, and MAC252 scFv proteins appeared to be due to the nature of those particular scFv genes. Investigation indicated that the single Vl domain of MAC61 limited the expression level mE. coli. The single Vl domain of MAC61 was expressed at least 10 fold lower than the single Vh domain. While a hybrid scFv gene which contained the Vl domain of MAC61 and the Vh domain of a readily expressed scFv was found to express at a much lower level than the hybrid scFv gene comprised of the Vl domain of a readily expressed scFv and the Vh domain of MAC61. Future experiments involving mutations of the amino acid sequence of the Vl domain could allow precise identification of the characteristics of the Vl domain of MAC61 which are responsible for the adverse effects on the expression in bacteria, yeast and plants.