Characterization of the glutamine synthetase amplifiable eukaryotic expression system applied to an integral membrane protein--the human thyrotropin receptor.
An amplifiable eukaryotic expression system, based upon glutamine synthetase, has been applied to the production of a complex integral membrane glycoprotein, the human receptor for the polypeptide hormone thyrotropin (TSH). Production of recombinant protein was achieved in chinese hamster ovary (CHO) cells at levels at least 10-fold higher than has been achieved in any other system. After amplification of the inserted gene, the gene copy number was found to be increased in most (but not all) subclones in the range of 3- to 50-fold; mRNA levels of the individual cell lines broadly followed their gene copy number. The level of protein production (measured both functionally and structurally, by radioligand binding and cytofluorimetry, respectively) also reflected these increases in DNA and RNA, but appeared to be limited to a maximum value which we conclude is the maximum that the cells can tolerate without impairing their viability. The receptor is efficiently coupled to adenylate cyclase (22-45 pM TSH producing a 50% response), although the coupling mechanism appeared to be saturated at higher receptor numbers. The high level of expression has allowed, for the first time, the detection of recombinant TSH receptor by immunochemical means. This expression system should prove very useful, not only in facilitating characterization of the TSH receptor, but also for the production of many other integral membrane proteins in their native form.
D000262:Adenylyl Cyclases / Q000378:metabolism / Q000502:physiology; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology*; D002999:Clone Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000032:analysis / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D008717:Methionine Sulfoximine / Q000494:pharmacology; D021141:Nucleic Acid Amplification Techniques; D012313:RNA / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D008717:Methionine Sulfoximine; D012313:RNA; D004247:DNA; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D005974:Glutamate-Ammonia Ligase