Approaches to maximizing stable expression of alpha 1-antitrypsin in transformed CHO cells.
A variety of approaches to maximizing the production of recombinant human alpha 1-antitrypsin (AAT) in Chinese hamster ovary (CHO) cells have been investigated. The highly active and inducible human cytomegalovirus immediate early (IE) promoter/enhancer was used to drive transcription of a recombinant AAT gene in transiently transfected and stably transformed CHO cells. The AAT gene was modified to incorporate highly efficient 3'RNA processing signals from the herpes simplex virus type 2 IE gene 5, and optimal translational initiation signals were created by site-directed mutagenesis. The effect of flanking the recombinant gene with matrix attachment regions was investigated. Combinations of these modifications allowed secretion of up to 44 micrograms AAT/ml per day by cell lines growing in serum-rich medium. This could be increased to up to 100 micrograms AAT/ml per day upon chemical induction of expression by propionate, butyrate or hexamethylene bisacetamide. Cell lines adapted to grow in protein-free medium produced less AAT but still responded to chemical induction to secrete up to 14 micrograms/ml per day of readily purified AAT.
Paterson
T
T
National Science Laboratory, Scottish National Blood Transfusion Service, Edinburgh, UK.
Innes
J
J
Moore
S
S
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002461:Cell Line, Transformed; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011422:Propionates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000235:genetics*
D002087:Butyrates; D011422:Propionates; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D020148:Butyric Acid; D004247:DNA; C029658:propionic acid; D004121:Dimethyl Sulfoxide