The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain.
A gene fragment encoding the extracellular domain of the human growth hormone (hGH) receptor from liver was cloned into a plasmid under control of the Escherichia coli alkaline phosphatase promoter and the heat-stable enterotoxin (StII) signal peptide sequence. Strains of E. coli expressing properly folded hGH binding protein were identified by blotting colonies with 125I-hGH. The E. coli strain capable of highest expression (KS330) secreted 10 to 20 mg/liter of culture of properly processed and folded hGH receptor fragment into the periplasmic space. The protein was purified to near homogeneity in 70 to 80% yield (in tens of milligram amounts) using ammonium sulfate precipitation, hGH affinity chromatography, and gel filtration. The unglycosylated extracellular domain of the hGH receptor has virtually identical binding properties compared to its natural glycosylated counterpart isolated from human serum, suggesting glycosylation is not important for binding of hGH. The extracellular binding domain codes for 7 cysteines, and we show that six of them form three disulfide bonds. Peptide mapping studies show these disulfides are paired sequentially to produce short loops (10-15 residues long) as follows: Cys38-Cys48, Cys83-Cys94, and Cys108-Cys122. Cys241 is unpaired, and mutagenic analysis shows that the extreme carboxyl end of the receptor fragment (including Cys241) is not essential for folding or binding of the protein to hGH. High level expression of this receptor binding domain and its homologs in E. coli will greatly facilitate their detailed biophysical and structural analysis.
Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
G
G
Fuh
M G
MG
Mulkerrin
S
S
Bass
N
N
McFarland
M
M
Brochier
J H
JH
Bourell
D R
DR
Light
J A
JA
Wells
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011986:Receptors, Somatotropin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004220:Disulfides; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D003545:Cysteine