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外文期刊>The biochemical journal
>Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis
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Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis
pSite-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDLsub3/sub [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.b3/b, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils iin vitro/i under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation./p
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