FIBRIL FORMATION FROM RECOMBINANT HUMAN SERUM AMYLOID-A

Three isotypes of human serum amyloid A (SAA), SAA1, SAA2 beta, and SA A4 were expressed at high levels in Escherichia coli (E. coli) using a pET vector expression system. Each SAA cDNA was ligated to the vector pET-21a(+) and transformed into E. coli, strain BL21(DE3)pLysS, Expre ssion conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to total E. coli proteins. E ach recombinant SAA (rSAA) was purified by molecular sieve chromatogra phy followed by chromatofocusing or hydrophobic interaction chromatogr aphy. The yield of purified protein was 5-10 mg per 11 of culture. Whe n subjected to in vitro fibril forming conditions, rSAA1 formed amyloi d-like fibrils confirmed by Congo red staining and electron microscopy . In contrast, rSAA2 beta and rSAA4 showed negative Congo red staining and curvilinear or flattened fibrillar structures on electron microsc opy. This suggests that SAA1 has greater potential for forming amyloid fibrils than either SAA2 beta or SAA4.