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 co mplementary 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 i n Escherichia coli, an intact full-length human cDNA. In this study, t he cDNA is shown to contain a cryptic bacterial promoter driving inapp ropriate expression of a part of human growth hormone (hGH) receptor w hich is toxic for E. coli growth. Identification of this promoter and its inactivation by changing only one nucleotide led us to obtain stab le bacterial clones containing a high copy number of full-length codin g sequences. This molecular clone was used in a baculovirus/insect cel l system to produce large amounts of glycosylated recombinant receptor . Binding studies with I-125-labelled hGH revealed an affinity constan t of 2.8.10(9) M-1, similar to that reported for the native liver rece ptor. This report described a general method of cloning which could be applied to similar unclonable cDNA fragments. (C) 1997 Elsevier Scien ce B.V.