Fibril formation from recombinant human serum amyloid A.
Fibril formation from recombinant human serum amyloid A.
复制标题
重组人血清淀粉样蛋白 A 形成原纤维。
DOI：
10.1016/0925-4439(94)90044-2
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Benson,MD
中科院分区：
文献类型：
--
作者：
Yamada,T;Kluve-Beckerman,B;Liepnieks,JJ;Benson,MD
利用pET载体系统，在大肠杆菌中高效表达了人血清淀粉样蛋白A的3个亚型：SAA1、SAA2、β和SAA4。将每一个SAA基因连接到载体pET-21a(+)上，转化E。ColiBL21(DE3)pLysS。表达条件需要高浓度的抗生素，以获得高比例的合成SAA和TOTALE。结肠蛋白。每个重组SAA(RSAA)用分子筛层析、聚焦层析或疏水作用层析纯化。纯化得率为5-10 mg/11。刚果红染色和电子显微镜证实，在体外纤维形成条件下，rSAA1形成了淀粉样原纤维。相反，rSAA2β和rSAA4在电子显微镜下显示刚果红染色阴性，纤维结构呈曲线或扁平。这表明，与SAA2、β或SAA4相比，SAA1形成淀粉样纤维的潜力更大。
Three isotypes of human serum amyloid A (SAA), SAA1, SAA2β, and SAA4 were expressed at high levels inEscherichia coli (E. coli)using a pET vector expression system. Each SAA cDNA was ligated to the vector pET-21a(+) and transformed intoE. coli, strain BL21(DE3)pLysS. Expression conditions required high concentrations of antibiotics in order to obtain a high ratio of synthesized SAA to totalE. coliproteins. Each recombinant SAA (rSAA) was purified by molecular sieve chromatography followed by chromatofocusing or hydrophobic interaction chromatography. The yield of purified 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, rSAA2β 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 SAA2β or SAA4.