Gardella J E, Gorgone G A, Candela L, Ghiso J, Castaño E M, Frangione B, Gorevic P D
Department of Medicine, State University of New York, Stony Brook 11794.
Biochem J. 1993 Sep 15;294 ( Pt 3)(Pt 3):667-74. doi: 10.1042/bj2940667.
We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.
我们使用设计用于便于克隆到T7表达载体pT7Ad23K13中的寡核苷酸引物，从cDNA中扩增编码阿尔茨海默病淀粉样前体蛋白（APP）3'端109个密码子的DNA，包括β蛋白（Aβ）和细胞质结构域。我们还通过定点诱变对该构建体进行修饰，以生成包含两个最近描述的APP突变体的重组分子。天然C109（包含APP C末端109个残基的缺失构建体）以及在密码子642处有单个突变（T→G，导致缬氨酸被甘氨酸取代）或在密码子595（G→T，赖氨酸被天冬酰胺取代）和596（A→C，甲硫氨酸被亮氨酸取代）处有双重突变的构建体，在大肠杆菌中诱导表达后，表达水平达到细菌总蛋白的5% - 20%。通过SDS/PAGE分析，表达的C109蛋白的主要成分表观分子量为16 - 18 kDa，从大小和N末端微测序来看似乎是全长构建体。还存在一种4 - 5 kDa的蛋白，它与C109共纯化，仅占表达蛋白的约1%，这通过用针对Aβ表位的特异性抗体进行Western印迹分析以及对纯化的重组C109进行生物素化后得以揭示。在生物合成标记实验中，该片段与全长C109共享N末端序列，似乎是由全长C109蛋白水解产生的。C109在针对NaCl或水进行透析后会自发沉淀，通过电子显微镜发现，长时间（> 20周）放置后，它含有少量（< 5%）与针对APP C末端表位的抗体发生反应的纤维状成分。重组C109似乎重现了在转染细胞、脑皮质和脑微血管中发现的APP C末端含Aβ片段的一些生化和物理化学特性。
