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 arranged in a planar configuration. In the present study a human CRP cDNA clone was ligated into the baculovirus vector pVL1393 which was used to establish a recombinant strain of BaculoGold Autographa californica multiple nuclear polyhedrosis virus containing the coding and leader sequence for human CRP (designated AcMNPV-CRP). Synthesis and secretion of CRP were studied after infection of TN5B1-4 and Sf-9 cells with AcMNPV-CRP. Accumulation of CRP but not other proteins in the medium over the course of infection suggested that CRP was actively secreted. Analysis by gel filtration chromatography and by SDS-PAGE demonstrated an intact pentamer composed of subunits of the appropriate molecular mobility. The structural integrity of the recombinant protein was further established by the ability of the product to bind to phosphocholine in a calcium-dependent manner, a property which is restricted to the intact pentamer. Functional studies of complement activation, binding to mononuclear phagocytic cells, and reactivity with a panel of monoclonal antibodies were also consistent with structural and functional integrity of the recombinant molecule. Infection of Trichoplusia ni larvae with AcMNPV-CRP also resulted in the production of functional recombinant protein. This method has the advantage of producing larger amounts of protein at lower cost than tissue culture. An additional advantage is the ability to metabolically label CRP through feeding the larvae on an [35S]methionine-containing diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Veterans Affairs Medical Center, Research Service, Albuquerque, NM 87108, USA.
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Marnell
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Mold
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Volzer
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Burlingame
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Du Clos
D000818:Animals; D002097:C-Reactive Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D007814:Larva / Q000235:genetics; D008970:Molecular Weight; D009036:Moths / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera; D013462:Sulfur Radioisotopes
D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D002097:C-Reactive Protein