Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7.
We made a mutated progastrin cDNA construct that contains a cleavage site (-Arg(-4)-Arg(-3)-Lys(-2)-Arg-1) specific for the Kex2-like endoprotease furin, located ahead of the bioactive gastrin. For expressing the mutated progastrin cDNA, we used two non-endocrine cell lines, CHO and COS-7. CHO cells exhibit amidating enzyme activity and levels of amidation enzyme mRNA as high as those in the pituitary-derived endocrine cell line GH3, whereas COS-7 cells have far less amidating activity and lower amounts of mRNA. Mutant progastrin-expressing CHO cells produced mostly amidated gastrin. Gel filtration showed the size of this gastrin corresponded to that of the synthetic human gastrin-17. In contrast, COS-7 cells produced glycine-extended gastrin and only a small amount of amidated gastrin. The difference in the amount of amidated gastrin products produced by the two non-endocrine cell lines is due to differing amounts of the amidation enzyme contained in each cell line.
Hayashi
N
N
Division of Molecular Endocrinology, Gunma University, Maebashi, Japan.
Kayo
T
T
Sugano
K
K
Takeuchi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D045683:Furin; D005755:Gastrins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016296:Mutagenesis; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013381:Subtilisins / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D005755:Gastrins; D009097:Multienzyme Complexes; D011498:Protein Precursors; D012333:RNA, Messenger; C013706:big gastrin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D013381:Subtilisins; D045683:Furin