High-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system.
A chemically synthesized gene (hGHR-ED) coding for the extracellular domain (ED) of the human growth hormone (hGH) receptor (hGHR) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. The secreted 35-kDa protein was purified to near homogeneity. The purified protein exhibited a high binding affinity (Kd = 0.2-0.3 nM) to hGH. The highest cell production capability was estimated at more than 10-20 micrograms hGHR-ED/ml of culture. The inhibition of the hGHR-ED secretion by treatment with tunicamycin suggests that glycosylation is important for secretion.
Protein Engineering Research Institute, Osaka, Japan.
Y
Y
Ota
A
A
Asakura
Y
Y
Matsuura
H
H
Kondo
A
A
Hitoshio
A
A
Iwane
T
T
Tanaka
M
M
Kikuchi
M
M
Ikehara
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D005813:Genes, Synthetic / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D000077183:Occlusion Body Matrix Proteins; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D011986:Receptors, Somatotropin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D010446:Peptide Fragments; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D014415:Tunicamycin