HIGH-LEVEL PRODUCTION OF HUMAN PARATHYROID-HORMONE IN BOMBYX-MORI LARVAE AND BMN CELLS USING RECOMBINANT BACULOVIRUS

A full-length cDNA encoding human parathyroid hormone (hPTH) containin g the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. Afte r transfection and generation of the recombinant baculovirus, hPTH pro duction was examined in silkworm larvae and BmN cell cultures. The lar vae synthesized and efficiently secreted the correctly processed and a uthentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN c ells, the major secreted form was the correctly sized protein, but sma ll amounts of degraded hPTH could also be detected in the medium by im munoblotting. Unlike the situation in larvae, prepro-hPTH could also b e demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 mu g/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected t o chemical and biological analyses. The authenticity of the purified r e-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone sho wed high-affinity receptor binding and full biological potency in incr easing cellular cAMP.