Open AccessHigh‐level Expression of the Human Neurokinin‐1 Receptor in Mammalian Cell Lines using the Semliki Forest Virus Expression System
Author(s) -
Lundström Kenneth,
Mills Ann,
Buell Gary,
Allet Elisabeth,
Adami Nadia,
Liljeström Peter
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00917.x
Subject(s) - semliki forest virus , chinese hamster ovary cell , biology , tachykinin receptor 1 , baby hamster kidney cell , receptor , microbiology and biotechnology , hamster , virology , virus , cell culture , rna , biochemistry , gene , substance p , genetics , neuropeptide
Human neurokinin‐1 receptor cDNA was introduced into the pSFV1 Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electroporated into BHK cells with pSFV‐Helper RNA. This procedure resulted in the packaging of a high‐titer SFV‐NK‐1 virus stock containing approximately 5×10 9 infective units/ml. Infection of baby hamster kidney, COS‐7, Chinese hamster ovary and human osteosarcoma cells yielded high levels of human neurokinin‐1 receptor expression as assessed by [ 3 H]substance P binding. The maximal receptor expression level obtained was 4×10 6 receptors/cell and studies of the post‐infection time indicated that a high level of receptor expression was observed 10–24 h post‐infection. The human neurokinin‐1 receptor expressed in infected baby hamster kidney, COS‐7 and Chinese hamster ovary cells was able to stimulate Ca 2+ mobilization indicating functional coupling to guanine‐nucleotide‐binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of large quantities of receptor protein for both pharmacological and structural studies.