Expression and characterization of recombinant human angiotensinogen in a heterologous eukaryotic cell line.
Transfection of Chinese hamster ovary cells with an expression plasmid containing a full length human angiotensinogen cDNA has provided cell lines that secrete recombinant angiotensinogen in large quantities. This angiotensinogen is immunologically identical to plasma angiotensinogen and can be cleaved by human kidney renin (EC 3.4.23.15.). The peptide liberated by renin cleavage is immunologically identical to standard angiotensin I and shows a retention time on isocratic reversed-phase high-pressure liquid chromatography identical to that of standard angiotensin I. The heterogeneity of recombinant angiotensinogen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis differs from that of plasma angiotensinogen. Treatment with endoglycosidases demonstrated that this difference is restricted to that of N-glycans and that N-glycans correspond to the quasi-totality of the carbohydrate content of both recombinant and plasma angiotensinogens. The development of a system capable of expressing human angiotensinogen cDNA in mammalian cells and the ability to obtain the corresponding angiotensinogen in large quantities will allow new studies on structure-function relationships of this protein.
Wei
L
L
INSERM U.36-17, Paris, France.
Gaillard
I
I
Corvol
P
P
Clauser
E
E
D000808:Angiotensinogen / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D005260:Female; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection
D011994:Recombinant Proteins; D000808:Angiotensinogen; D004247:DNA