Recombinant murine serum amyloid A from baculovirus-infected insect cells: purification and characterization.
Serum amyloid A (SAA) is an extremely sensitive acute-phase reactant and precursor to the subunit protein in reactive amyloid deposits. Although the mouse has long served as an informative experimental model, both the function of SAA and the pathogenic mechanism of amyloid formation remain unknown. The production of SAA by a heterologous system was pursued as means of generating readily-renewable amounts of SAA of defined sequence. Murine SAA2 has been expressed in and purified from baculovirus-infected insect cells. Using the transfer vector pBlueBac, SAA2 cDNA was cloned into baculovirus DNA such that expression was under the control of the polyhedrin promoter. Lysates prepared from infected cells contained three amyloid A-immunoreactive forms which accumulated intracellularly over a three day periods. The form having the lowest relative molecular mass, 12.5 kDa, co-migrated in SDS-polyacrylamide gels with the SAA2 present in murine acute-phase serum. Recombinant SAA2 was purified by Sepharose CL-6B chromatography followed by chromatofocusing between pH 8 and pH 5. Amino-terminal sequencing of the purified 12.5 kDa sample confirmed the first 20 residues of mature murine SAA2. After incubation with normal mouse serum, purified recombinant SAA2 fractionated exclusively with lipoprotein complexes, suggesting that it was bound to HDL. Based on this observation, we believe that recombinant SAA can serve as a suitable substitute for the native protein in physiologically relevant studies.
Kluve-Beckerman
B
B
Department of Medicine, Indiana University School of Medicine, Indianapolis.
Song
M
M
Benson
M D
MD
Liepnieks
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D000685:Serum Amyloid A Protein / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; C489995:Saa2 protein, mouse; D000685:Serum Amyloid A Protein