Human growth hormone receptor: cloning and expression of the full-length complementary DNA after site-directed inactivation of a cryptic bacterial promoter.
Growth hormone receptor is a cytokine-type receptor which is required for normal somatic growth and for numerous metabolic processes. Its complementary DNA (cDNA) has been isolated in various species leading to intensive studies to elucidate the mechanism of action of the growth hormone. However, serious difficulties have been reported in cloning in Escherichia coli, an intact full-length human cDNA. In this study, the cDNA is shown to contain a cryptic bacterial promoter driving inappropriate expression of a part of human growth hormone (hGH) receptor which is toxic for E. coli growth. Identification of this promoter and its inactivation by changing only one nucleotide led us to obtain stable bacterial clones containing a high copy number of full-length coding sequences. This molecular clone was used in a baculovirus/insect cell system to produce large amounts of glycosylated recombinant receptor. Binding studies with 125I-labelled hGH revealed an affinity constant of 2.8 x 10(9) M(-1), similar to that reported for the native liver receptor. This report described a general method of cloning which could be applied to similar unclonable cDNA fragments.
Bieth
E
E
Centre de Recherche sur la Croissance, Pavillon Leriche, Centre Hospitalier Universitaire de Purpan, Toulouse, France.
Cahoreau
C
C
Cholin
S
S
Molinas
C
C
Cerutti
Rochiccioli
P
P
Devauchelle
G
G
Tauber
M
M
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006031:Glycosylation; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011986:Receptors, Somatotropin / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D015183:Restriction Mapping; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D019382:Human Growth Hormone