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 pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation 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 and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM L-arginine, 2 mM oxidized glutathione 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 renatured. Juvenile black sea bream injected with 0.05 microgram/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system.
Hsih
M H
MH
Institute of Fisheries Science, National Taiwan University, Taipei.
Kuo
J C
JC
Tsai
H J
HJ
D000818:Animals; D017756:Aquaculture; D001120:Arginine / Q000494:pharmacology; D001681:Biological Assay; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002479:Inclusion Bodies; D010473:Perciformes / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility
D011994:Recombinant Proteins; D013006:Growth Hormone; D001120:Arginine; D005978:Glutathione; D019803:Glutathione Disulfide