Large-scale purification and refolding of recombinant eel growth hormone.

In the past decade, growth hormones (GHs) have been isolated from the pituitaries of several teleost species including tilapia, salmon, carp, and eel.1~9) Although teleost GHs may be useful in promoting growth in aquaculture, little is known about appropriate routes, doses, or patterns of administration. Considerable amounts of teleost GHsare required to clarify both the mode of action of teleost GHand the mechanisms that control its secretion. In contrast with mammals, in which preparations of recombinant GHs are readily available,10'11} purification of a teleost GHhas yet to be reported. Two forms of GH (EGH I and II) have been isolated from eel pituitaries and their primary structures have been analyzed by Kishida et al.9) and Yamaguchi et al.;12) EGH II lacks three amino acids at the N-terminal of EGHI. Molecular cloning of EGH CDNAand its expression in Escherichia coli (E. coli) have been done by Saito et al.13) In this study, we have established a procedure for large-scale preparation of recombinant EGH II (rEGH) and confirmed its electrophoretic, immunological, and biological identities with natural EGHs derived from eel pituitaries. High-level expression of rEGH in E. coli was achieved in a 5-1 fermentor.13) E. coli cells containing rEGH inclusion bodies were harvested from 31 of broth, suspended in 0.4 1 of 20mM phosphate buffer (pH 7.2), and broken by passage through a Manton Gaulin homogenizer. The resulting homogenate was centrifuged at 8000rpm for 40min at 4°C, and the pellet was washed with 0.41 of 1 m sucrose and then with 0.2 1 of4% Triton X-100, 20mMphosphate buffer, and 1 mMEDTA (pH 7.2) to purify inclusion bodies of rEGH. Soluble components, bacterial cell wall and cell membrane, and lipid components were removed by these processes. The