Ž . Molecular Brain Research 44 1997 143–146 Short communication A recombinant adenovirus that directs secretion of biologically active k-bungarotoxin from mammalian cells S. Gorman a , N. Viseshakul b , B. Cohen b , S. Hardy c , G.A. Grant d , C.S. Yost a , J.R. Forsayeth a, ) a Department of Anesthesia, UniÕersity of California San Francisco, San Francisco, CA 94143-0542, USA b DiÕision of Biomedical Sciences, UniÕersity of California RiÕerside, RiÕerside, CA 92521-0121, USA c Somatix Therapy Corporation, Alameda, CA 94501, USA d Department of Molecular Biology and Pharmacology, Washington UniÕersity, St. Louis, MO 63110, USA Accepted 24 September 1996 Abstract Ž . A novel Cre-lox system was used to construct an adenovirus encoding k-bungarotoxin k-Bgt , modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus Ž . demonstrated that recombinant k-Bgt R-k-Bgt was secreted. The biological activity of the secreted R-k-Bgt was investigated in Ž . Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor nAChR subtypes a 3 b 2 and a 2 b 2. The recombinant toxin inhibited the response of a 3 b 2 type AChRs to ACh, but did not inhibit the response of a 2 b 2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active k-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible. Keywords: Neuronal; k-Bungarotoxin; Adenovirus; Recombinant; Acetylcholine; Nicotinic; Oocyte Toxins derived from venoms have become important tools in biological research, and particularly so in neuro- science. Perhaps the most widely used are the bungarotox- ins, derived from the venom of the banded krait, Bungarus wx multicinctus 3. One of the bungarotoxins is a- Ž . bungarotoxin a-BTX , which binds with remarkable affinity to the muscle type nicotinic acetylcholine receptor Ž . AChR , thereby blocking activation of the receptor by acetylcholine and inducing muscle paralysis. Its very slow rate of dissociation from the AChR makes a-BTX an excellent ligand for labeling, for affinity purification, and for pharmacological analysis, of the AChR. Banded krait venom, from which a-BTX is purified, also contains another toxin, variously named toxin 3.1, toxin F, neuronal bungarotoxin, or k-bungarotoxin. This k-bungarotoxin shares homology at the amino acid level with a-BTX but binds to a different class of AChR expressed primarily in w x the peripheral nervous system 9–11 . Molecular cloning ) Ž . Corresponding author. Fax: q1 415 476-8841. of neuronal AChR genes has generated an increased need for tools to study this class of receptors. One difficulty in using k-Bgt has been its low abun- dance in venom, and the unavailability of the now pro- tected Bungarus multicinctus. Previous efforts to produce recombinant k-Bgt from a cDNA in bacteria resulted in a wx low yield of active toxin 7 . Although expression of k-Bgt wx in the Picchia yeast strain 8 has resulted in greatly improved yields of active toxin, a problem with using it in vivo is that it is very difficult to deliver the toxin at a maintained level to tissues over a period of time. Aden- oviruses can be used to express gene products directly in mammalian tissues and expression is stable over time in wx the CNS 1 . Here we report the construction of an aden- ovirus that encodes a secreted form of active k-Bgt. k-Bgt contains 10 cysteines of a total of 66 amino acids w x 5,9 . Bacteria do not catalyze the formation of disulfide bonds efficiently. However, in the lumen of the endoplas- mic reticulum disulfide bond formation is favored. For this wx X reason, k-Bgt cDNA 7 was attached to the 3 end of the w x bovine prolactin signal sequence by assembly PCR 16 . 0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 96 00250-1