Expression and purification of soluble, active heterodimeric guanylyl cyclase from baculovirus.
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.
Gupta
G
G
Columbia University, College of Physicians & Surgeons, New York, New York 10032, USA.
Kim
J
J
Yang
L
L
Sturley
S L
SL
Danziger
R S
RS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D007527:Isoenzymes / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011524:Protoporphyrins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera
D007527:Isoenzymes; D011524:Protoporphyrins; D011994:Recombinant Proteins; D006639:Histidine; D006162:Guanylate Cyclase