RECOMBINANT MURINE SERUM AMYLOID-A FROM BACULOVIRUS-INFECTED INSECT CELLS - PURIFICATION AND CHARACTERIZATION

Serum amyloid A (SAA) is an extremely sensitive acute-phase reactant a nd precursor to the subunit protein in reactive amyloid deposits. Alth ough the mouse has long served as an informative experimental model, b oth the function of SAA and the pathogenic mechanism of amyloid format ion remain unknown. The production of SAA by a heterologous system was pursued as means of generating readily-renewable amounts of SAA of de fined sequence. Murine SAA2 has been expressed in and purified from ba culovirus-infected insect cells. Using the transfer vector pBlueBac, S AA2 cDNA was cloned into baculovirus DNA such that expression was unde r the control of the polyhedrin promoter. Lysates prepared from infect ed cells contained three amyloid A-immunoreactive forms which accumula ted intracellularly over a three day period. The form having the lowes t relative molecular mass, 12.5 kDa, co-migrated in SDS-polyacrylamide gels with the SAA2 present in murine acute-phase serum. Recombinant S AA2 was purified by Sepharose CL-6B chromatography followed by chromat ofocusing between pH 8 and pH 5. Amino-terminal sequencing of the puri fied 12.5 kDa sample confirmed the first 20 residues of mature murine SAA2. After incubation with normal mouse serum, purified recombinant S AA2 fractionated exclusively with lipoprotein complexes, suggesting th at it was bound to HDL. Based on this observation, we believe that rec ombinant SAA can serve as a suitable substitute for the native protein in physiologically relevant studies.