|Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
1. Purification of human prolactin (hPRL), human growth hormone (hGH) and flounder growth hormone (fGH) expressed in E. coli.
(1) Expression vectors for hPRL, hGH and fGH were constructed using Taq promotor and each cDNA of hPRL, hGH and fGH, and were introduced into E. coli. The transformed E. coli expressed large amouamount of each protein, about 20% of the total E. coli proteins.
(2) The recombinant hormones were extracted from the E. coli cells, refolded with gultathione, and were purified to homogeneity by gel filtration and anion exchange chromatography.
2. Analysis of biological activities of recombinant PRL and GHs
(1) Biological activities of the recombinant hPRL and hGH were assayed for ability to stimulate the growth of Nb2 lymphoma cells and for ability to induce N-acetyllactosamine synthase in mammary grand cells, respectively. The biological activities of both recombinant hormones were equivalent to those of the corresponding native hormones.
(2) Biological activity of the recombinant fGH was assayed for growth promoting activity for rainbow trout. Although the fGH lacks the region composing 13-14 amino acid residues near C-terminal in other GHs including yellow tail GH, the recombinant fGH has exhibited equal activity to a recombinant yellow tail GH. This suggests that the region lacking in the fGH is not essential for the growth promoting activity.
3. Analysis of primary structure of prolactin-like hormones in bovine placenta. Four novel cDNA clones for prolactin-like proteins, bPLP-I, bPLP-II, bPLP-III and bPLP-IV were isolated from a bovine placental cDNA library, and their complete nucleotide sequences were determined. One of the clones, bPLP-IV, lacks the two C-terminal cysteine residues which are completely conserved in all the known members of GH/PRL gene family, suggesting that the two C-terminal cysteine residues may not be essential for lactogenic activity.