Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese

David M. Miller; Gary P. Kurzban; Jose A. Mendoza; John M. Chirgwin; Stephen C. Hardies; Paul M. Horowitz

doi:10.1016/0167-4838(92)90158-A

Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese

David M. Miller, Gary P. Kurzban, Jose A. Mendoza, John M. Chirgwin, Stephen C. Hardies, Paul M. Horowitz

Research output: Contribution to journal › Article › peer-review

34 Scopus citations

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 K_m 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	https://doi.org/10.1016/0167-4838(92)90158-A
State	Published - Jun 24 1992
Externally published	Yes

Keywords

(E. coli)
Bovine liver rhodanese
Chaperonin
Protein expression
Protein folding
Protein purification
Recombinant rhodanase

ASJC Scopus subject areas

Structural Biology
Biophysics
Biochemistry
Molecular Biology

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10.1016/0167-4838(92)90158-A

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@article{413162ba9df04e5e8757d12462490a24,

title = "Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese",

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.",

keywords = "(E. coli), Bovine liver rhodanese, Chaperonin, Protein expression, Protein folding, Protein purification, Recombinant rhodanase",

author = "Miller, {David M.} and Kurzban, {Gary P.} and Mendoza, {Jose A.} and Chirgwin, {John M.} and Hardies, {Stephen C.} and Horowitz, {Paul M.}",

note = "Funding Information: This work was supported by research grants GM25177 and ES05729f rom the National Instituteso f Health and AQ-723 from the Robert A. Welch Foundation to PMH, Grants HG00190 from the National Instituteso f Health and AQ-1107 from the Robert A. Welch Foundation( to SCH) and Grant AQ-1123 from the Robert A. Welch Foundationa nd a Veterans Ad-ministrationm erit award (to JMC). GPK is a Robert A. Welch Fellow. We thank Peggy Rifleman of the BiopolymerS equencinga nd SynthesisF acility, University of Texas Health Science Center at San Antonio, for sequencingt he amino terminus of recombinant rhodanese.",

year = "1992",

month = jun,

day = "24",

doi = "10.1016/0167-4838(92)90158-A",

language = "English (US)",

volume = "1121",

pages = "286--292",

journal = "BBA - Protein Structure",

issn = "1570-9639",

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number = "3",

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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.

N1 - Funding Information: This work was supported by research grants GM25177 and ES05729f rom the National Instituteso f Health and AQ-723 from the Robert A. Welch Foundation to PMH, Grants HG00190 from the National Instituteso f Health and AQ-1107 from the Robert A. Welch Foundation( to SCH) and Grant AQ-1123 from the Robert A. Welch Foundationa nd a Veterans Ad-ministrationm erit award (to JMC). GPK is a Robert A. Welch Fellow. We thank Peggy Rifleman of the BiopolymerS equencinga nd SynthesisF acility, University of Texas Health Science Center at San Antonio, for sequencingt he amino terminus of recombinant rhodanese.

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

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Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese

Abstract

Keywords

ASJC Scopus subject areas

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