Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells

Protein Expression and Purification
Andre B H ChooR L Raison

Abstract

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/10(4) cells. Cytotoxicity...Continue Reading

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Citations

May 20, 2005·Gene Therapy·L Johannes, D Decaudin
Oct 10, 2006·The AAPS Journal·Robert J Kreitman
Apr 7, 2009·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Robert J Kreitman
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Jun 7, 2003·Parasite Immunology·Francisco J Alarcon-ChaidezStephen Wikel
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Feb 15, 2021·Biologicals : Journal of the International Association of Biological Standardization·Jie TaoHuili Liu
Aug 27, 2003·Seminars in Oncology·Arthur E FrankelStephen H Leppla

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