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) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/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 to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.
Mathavan
S
S
School of Biological Sciences, Madurai Kamaraj University, India.
Gautvik
V T
VT
Rokkones
E
E
Olstad
O K
OK
Kareem
B N
BN
Maeda
S
S
Gautvik
K M
KM
D000818:Animals; D016367:Baculoviridae; D012831:Bombyx / Q000235:genetics; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006458:Hemolymph / Q000166:cytology; D006801:Humans; D007814:Larva; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D010281:Parathyroid Hormone; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000242:Cyclic AMP