OPTIMIZATION OF THE SOLUBILIZATION AND RENATURATION OF FISH GROWTH-HORMONE PRODUCED BY ESCHERICHIA-COLI

Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitar y glands, However,fish recombinant GH (rGH) can be efficiently synthes ized by Escherichia coli cells, although it exists in denatured form i n inclusion bodies (IB). We studied the solubilization of IB and the r enaturation of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 +/- 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl beta-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM beta-lactose, 95.3 +/- 3.43 mg of IB was obtained, The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride a nd then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbo nate and 2 mM EDTA) containing 100 mM L-arginine, 2 mM oxidized glutat hione and 2 mM reduced glutathione for 24 h at 4 degrees C in a volume ratio of 3 to 500, At least 20% of the denaturated rGH in IB was rena tured. Juvenile black sea bream injected with 0.05 mu g/g resultant rG H once every 2 weeks exhibited significant increases (P < 0.05) in wei ght gain (84%) relative to fish in the control group over a 16-week pe riod. This process is an economical and effective way to obtain an act ive form of rGH biosynthesized by a prokaryotic system.