title: Fibril formation from recombinant human serum amyloid A.
abstract: Three isotypes of human serum amyloid A (SAA), SAA1, SAA2 beta, and SAA4 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. Expression conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to total E. coli proteins. Each recombinant SAA (rSAA) was purified by molecular sieve chromatography followed by chromatofocusing or hydrophobic interaction chromatography. The yield of purified protein was 5-10 mg per 11 of culture. When subjected to in vitro fibril forming conditions, rSAA1 formed amyloid-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 microscopy. This suggests that SAA1 has greater potential for forming amyloid fibrils than either SAA2 beta or SAA4.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis.
authors.forename: T
authors.initials: T
authors.lastname: Yamada
authors.forename: B
authors.initials: B
authors.lastname: Kluve-Beckerman
authors.forename: J J
authors.initials: JJ
authors.lastname: Liepnieks
authors.forename: M D
authors.initials: MD
authors.lastname: Benson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001718:Birefringence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D000685:Serum Amyloid A Protein / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D000685:Serum Amyloid A Protein