title: Synthesis and purification of a toxin-linked conjugate targeting epidermal growth factor receptor in Escherichia coli.
abstract: Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5 kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni(2+)-affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.
authors.affiliation: Department of Neurosurgery, The First Hospital of Jilin University, Changchun, Jilin, PR China.
authors.forename: Chengyuan
authors.initials: C
authors.lastname: Ma
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Li
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.forename: Kan
authors.initials: K
authors.lastname: Xu
authors.forename: Haiyang
authors.initials: H
authors.lastname: Xu
authors.forename: Jieying
authors.initials: J
authors.lastname: Bai
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005434:Flow Cytometry; D005909:Glioblastoma / Q000201:enzymology; D006801:Humans; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054788:Ribosome Inactivating Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C512478:EGFR protein, human; D066246:ErbB Receptors; D054788:Ribosome Inactivating Proteins