Soluble Expression of a Functional Recombinant Cytolytic Immunotoxin in Insect Cells

• Published in: Protein expression and purification 2002-04, Vol.24 (3), p.338-347
• Main Authors: Choo, Andre B.H., Dunn, Rosanne D., Broady, Kevin W., Raison, Robert L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Fluorescent Antibody Technique; Genetic Vectors; Glycoproteins - genetics; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - metabolism; Immunotoxins - genetics; Immunotoxins - metabolism; Melitten - genetics; Melitten - metabolism; Melitten - toxicity; Molecular Sequence Data; Oligopeptides; Peptides - genetics; Peptides - metabolism; Peptides - toxicity; Protein Sorting Signals - genetics; Spodoptera
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2001.1589

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3–5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/104 cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.