Characterization of an associated microfibril protein through recombinant DNA techniques.
Characterization of an associated microfibril protein through recombinant DNA techniques.
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DOI：
10.1016/s0021-9258(19)50203-4
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发表时间：
1992-05
期刊：
影响因子：
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通讯作者：
S. Horrigan;C. Rich;Barbara W. Streeten;Zong-Yi Li;Judith Ann Foster
中科院分区：
文献类型：
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作者：
S. Horrigan;C. Rich;Barbara W. Streeten;Zong-Yi Li;Judith Ann Foster
通过重组 DNA 技术确定了与弹性纤维微原纤维相关的新型细胞外蛋白的完整一级结构。使用针对不溶性牛眼小带蛋白的抗血清来筛选由整个鸡胚poly(A)+RNA构建的lambda gt11 cDNA表达文库。然后使用编码免疫反应性融合多肽的cDNA通过噬斑杂交重新筛选文库。重叠 cDNA 克隆的核苷酸测序揭示了一个 1326 个碱基的开放翻译阅读框，从起始起始序列开始，到终止密码子结束。连续的 cDNA 序列包含一个 563 个碱基的 3'-非翻译区，在 Poly(A) 尾上游 16 个碱基处可能有一个聚腺苷酸化位点。鸡主动脉 mRNA 的引物延伸与序列数据结合起来，揭示了从翻译起始位点向上游延伸的 95 个碱基的 5'-非翻译区域。 Northern 印迹分析表明，分离的 cDNA 与整个鸡胚以及鸡胚主动脉、心脏和肌肉 RNA 制剂中的 2.1 KB mRNA 杂交。 cDNA编码的初始翻译蛋白为53,932 kDa，具有亲水性氨基酸组成，其中谷氨酸占总氨基酸残基的22%。针对推断的蛋白质一级结构中编码的合成肽序列（14 个氨基酸）引发抗血清。在 β-氨基丙腈存在下培养的鸡胚主动脉中提取的蛋白质的蛋白质印迹显示，培养基和温和的盐提取物含有表观分子量为 58,000 的免疫反应性蛋白质，而严酷的变性剂则提取出 32,000 kDa 的蛋白质。使用放射性标记赖氨酸的脉冲追踪实验表明，新合成的 58,000 kDa 蛋白质在 2-24 小时内被追踪为 32,000 kDa 蛋白质。对鸡主动脉、牛颈韧带和人眼小带的组织切片进行免疫电镜检查，结果表明肽引发的抗体特异性定位于超微结构可定义的微纤维结构。
The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3‘-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5‘-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.