EXPRESSION AND PURIFICATION OF SOLUBLE, ACTIVE HETERODIMERIC GUANYLYLCYCLASE FROM BACULOVIRUS

A method for expression and purification of active cytosolic heterodim eric histidine (His)-tagged guanylyl cyclase of the alpha 1/beta 1 iso form has been developed using recombinant baculovirus-transfected inse ct cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-t erminus of the alpha 1 and beta 1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity ste p using a nickel column. A second gel-filtration step was applied to r econstitute into the complex heme, a required cofactor. This was confi rmed 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-proto porphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic g uanylyl cyclase and facilitate mechanistic and structural studies of t his important family of enzymes. Furthermore, the procedure demonstrat es the utility of the His-tag system to purify multimeric proteins. (C ) 1997 Academic Press.