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A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the alpha 1/beta 1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the alpha 1 and beta 1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
September 2000,
Proceedings of the National Academy of Sciences of the United States of America,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
January 1991,
Methods in enzymology,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
January 1991,
Methods in enzymology,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
January 2000,
Nihon rinsho. Japanese journal of clinical medicine,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
March 1998,
Archives of biochemistry and biophysics,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
January 1991,
Proceedings of the National Academy of Sciences of the United States of America,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
July 1998,
Naunyn-Schmiedeberg's archives of pharmacology,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
June 1994,
The Journal of biological chemistry,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
June 1990,
European journal of biochemistry,
G Gupta, and
J Kim, and
L Yang, and
S L Sturley, and
R S Danziger
December 1991,
Biochimica et biophysica acta,
