The generation of stable CHO cell lines expressing very high levels of complement C5A receptors and subsequent modulation of binding affinity for C5A.
A single gene fragment encoding for the C5a receptor was produced by reverse transcription of mRNA isolated from differentiated U937 cells and subsequently amplified by means of the polymerase chain reaction. This fragment was introduced into the mammalian expression vector pEE6hCMV.neo and used to transfect a CHO cell line constitutively expressing a viral transactivator protein. Binding characteristics identical to the native neutrophil receptor were observed. A combination of antibiotic selection and cell sorting using anti-C5a receptor antiserum were then used to generate stable cell lines expressing up to 1.2 x 10(7) functional C5a receptors/cell with a lower affinity for C5a. It is postulated that this modulation of receptor affinity is dependent on coupling to native G-proteins in the host cells.
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
J E
JE
Pease
D R
DR
Burton
M D
MD
Barker
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D015936:Complement C5a / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D004247:DNA