Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells.
Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In an in vivo bioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.
Zhang
J
J
Department of Protein Development, Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, Maryland 20850, USA. junli_zhang@hgsi.com
Alfonso
P
P
Thotakura
N R
NR
Su
J
J
Buergin
M
M
Parmelee
D
D
Collins
A W
AW
Oelkuct
M
M
Gaffney
S
S
Gentz
S
S
Radman
D P
DP
Wagner
G F
GF
Gentz
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001681:Biological Assay; D016466:CHO Cells / Q000821:virology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006728:Hormones / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D006023:Glycoproteins; D006728:Hormones; D011994:Recombinant Proteins; C051795:teleocalcin; D002118:Calcium