Expression and Purification of a Foreign Gene Delivery Fusion Protein anti-LMP1scFv/tP in Escherichia coli

Abstract: Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) constitutively expresses on cell surface of various cancer cells. In this study， we aim to construct a fusion protein that contains a domain of the single chain of the human variable fragment (scFv) against LMP1 and a nucleotide-binding domain of truncated protamine (tP). The fusion protein anti-LMP1scFv/tP was designed with the tP coding sequence linked to the 3´- terminus of the scFv against LMP1， which will not only target LMP1 but also maintain nucleotide binding activity. The anti-LMP1scFv/tP gene was obtained by PCR-based gene assembly. The fusion protein was expressed in inclusion bodies in Escherichia coli Rosseta and was purified efficiently by immobilized metal (Ni2+) affinity chromatography with His-tag under denaturation condition， and then the purified product was refolded by dialysis against urea concentration gradient. Indirect cellular immunofluorescence staining confirmed that refolded anti- LMP1scFv/tP maintained antigen binding activity， which could bind to CNE1-GL cells expressing LMP1 and couldn’t bind to CNE1 cells not expressing LMP1. Gel shift assay demonstrated that refolded anti-LMP1scFv/tP had DNA binding activity. Thus， the fusion protein provides a basis for further application for targeting gene delivery to LMP1 expressing tumor cells.

CSTR: 32200.14.cjcb.2010.03.0008