Biochemical characterization and solubilization of human NK2 receptor expressed in Chinese hamster ovary cells.
The human ileum neurokinin NK2 receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7 x 10(5) NK2 receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [125I]NKA to NK2 receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK2 receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK2 receptor protein from mammalian cells.
Turcatti
G
G
Glaxo Institute for Molecular Biology S.A., Geneve, Switzerland.
Ceszkowski
K
K
Chollet
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002793:Cholic Acids; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003902:Detergents; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015288:Neurokinin A / Q000378:metabolism*; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D015288:Neurokinin A; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate