Abstract
Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33 000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 286-292 |
| Number of pages | 7 |
| Journal | Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular |
| Volume | 1121 |
| Issue number | 3 |
| DOIs | |
| State | Published - Jun 24 1992 |
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Keywords
- (E. coli)
- Bovine liver rhodanese
- Chaperonin
- Protein expression
- Protein folding
- Protein purification
- Recombinant rhodanase
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
- Structural Biology
Cite this
Recombinant bovine rhodanese : purification and comparison with bovine liver rhodanese. / Miller, David M.; Kurzban, Gary P.; Mendoza, Jose A.; Chirgwin, John M.; Hardies, Stephen C.; Horowitz, Paul M.
In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1121, No. 3, 24.06.1992, p. 286-292.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Recombinant bovine rhodanese
T2 - purification and comparison with bovine liver rhodanese
AU - Miller, David M.
AU - Kurzban, Gary P.
AU - Mendoza, Jose A.
AU - Chirgwin, John M.
AU - Hardies, Stephen C.
AU - Horowitz, Paul M.
PY - 1992/6/24
Y1 - 1992/6/24
N2 - Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33 000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.
AB - Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33 000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.
KW - (E. coli)
KW - Bovine liver rhodanese
KW - Chaperonin
KW - Protein expression
KW - Protein folding
KW - Protein purification
KW - Recombinant rhodanase
UR - http://www.scopus.com/inward/record.url?scp=0026710673&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026710673&partnerID=8YFLogxK
U2 - 10.1016/0167-4838(92)90158-A
DO - 10.1016/0167-4838(92)90158-A
M3 - Article
C2 - 1627606
AN - SCOPUS:0026710673
VL - 1121
SP - 286
EP - 292
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
SN - 1570-9639
IS - 3
ER -