1. Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis
- Author
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H, Ogawa, F, Takusagawa, K, Wakaki, H, Kishi, M R, Eskandarian, M, Kobayashi, T, Date, N H, Huh, and H C, Pitot
- Subjects
Male ,L-Serine Dehydratase ,Protein Denaturation ,Protein Folding ,DNA, Complementary ,Hydrolysis ,Serine Endopeptidases ,Recombinant Proteins ,Rats ,Liver ,Pyridoxal Phosphate ,Escherichia coli ,Animals ,Trypsin ,Cloning, Molecular ,Rats, Wistar ,Protein Binding - Abstract
A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases.
- Published
- 1999