EXPRESSION AND RADIOLABELING OF HUMAN C-REACTIVE PROTEIN IN BACULOVIRUS-INFECTED CELL-LINES AND TRICHOPLUSIA NI LARVAE

Human C-reactive protein (CRP) is a member of the pentraxin family of proteins which are molecules composed of five identical subunits arran ged in a planar configuration. In the present study a human CRP cDNA c lone was ligated into the baculovirus vector pVL1393 which was used to establish a recombinant strain of BaculoGold Autographa californica m ultiple nuclear polyhedrosis virus containing the coding and leader se quence for human CRP (designated AcMNPV-CRP). Synthesis and secretion of CRP were studied after infection of TN5B1-4 and Sf-9 cells with AcM NPV-CRP, Accumulation of CRP but not other proteins in the medium over the course of infection suggested that CRP was actively secreted, Ana lysis by gel filtration chromatography and by SDS-PAGE demonstrated an intact pentamer composed of subunits of the appropriate molecular mob ility. The structural integrity of the recombinant protein was further established by the ability of the product to bind to phosphocholine i n a calcium-dependent manner, a property which is restricted to the in tact pentamer, Functional studies of complement activation, binding to mononuclear phagocytic cells, and reactivity with a panel of monoclon al antibodies were also consistent with structural and functional inte grity of the recombinant molecule. Infection of Trichoplusia ni larvae with AcMNPV-CRP also resulted in the production of functional recombi nant protein, This method has the advantage of producing larger amount s of protein at lower cost than tissue culture, An additional advantag e is the ability to metabolically label CRP through feeding the larvae on an [S-35]methionine-containing diet. The ability to produce suffic ient quantities of metabolically labeled recombinant protein for recep tor binding and structure-function studies of mutagenized molecules is a useful property of this system.