Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition - Studies with recombinant ovotransferrins
Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition - Studies with recombinant ovotransferrins
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DOI：
10.1042/bj3190361
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发表时间：
1996-10-15
影响因子：
4.1
通讯作者：
MacGillivray, RTA
中科院分区：
文献类型：
--
作者：
Mason, AB;Woodworth, RC;MacGillivray, RTA
用含有鸡卵转铁蛋白（oTF/2N）相关DNA编码序列的质粒pNUT转染幼仓鼠肾细胞，从其组织培养液中分离到不同的重组体oTF/2N。对于分别限定残基1-319、1-332和1-337-（Ala）的构建体，获得了高达40、55和30 mg/l oTF/2N的水平（3）。此外，全长非糖基化oTF以最大80 mg/l表达，由残基1-682组成的缩短的oTF以95 mg/l的水平表达。然后将这些制备物用于蛋白水解产生包含残基342-686和342-682的两种不同的C端半段（oTF/2C）。纯化的重组N-叶（oTF/2N）是类似的蛋白水解衍生的半分子的免疫反应性和光谱特性方面，他们表现出一些有趣的差异，热稳定性。cDNA的序列分析揭示了核苷酸水平上的六个变化，与Jeltsch和Chambon [（1982）Eur. J.Biochem.122，291-295]，电喷雾质谱给出与这六种变化一致的结果。通过滴定量热法测量各种N-和C-叶之间的相互作用。研究表明，只有那些在溶液中结合的叶才能够与鸡胚红细胞上的受体结合。这些发现并不支持Oratore等人先前的报告[（1989）Biochem.J.257，301-304]。
Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/l oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)(3) respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/l and a foreshortened oTF consisting of residues 1-682 was expressed al a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291-295], Electrospray mass spectrometry gives results consistent with these six changes, Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al. [(1989) Biochem. J. 257, 301-304].