Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro-Pro extension.

ABSTRACT

Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro-Pro-hGHRH(1-44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro-pro-hGHRH(1-44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp-Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS-PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 microg/ml used, respectively, released the GH values of 0.1+/-0.1, 12.5+/-7.3, 16.6+/-5.8, 49.8+/-7.6, and 79.5+/-5.7 ng/ml whereas their blank controls, respectively, were 0.5+/-0.8, 4.1+/-2.6, 3.1+/-3.1, 4.7+/-1.8, and 1.2+/-0.3 ng/ml. The activity results of all dose groups except 0.01 microg/ml Pro-Pro-hGHRH(1-44) group and hGHRH(1-40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1-40) showed no measured GH release when the dose was up to 2 microg/ml. The activity results show that the Pro-Pro-hGHRH(1-44) peptide is a potential GH releasing analog.