High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli.
High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli.
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DOI：
10.1111/j.1432-1033.1995.0788h.x
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发表时间：
1995-06
期刊：
影响因子：
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通讯作者：
N. Budisa;Boris Steipe;P. Demange;C. Eckerskorn;J. Kellermann;R. Huber
中科院分区：
文献类型：
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作者：
N. Budisa;Boris Steipe;P. Demange;C. Eckerskorn;J. Kellermann;R. Huber
我们已经利用了T7聚合酶/启动子系统的高水平纳入蛋氨酸类似物与合适的标签的结构研究（X-射线和NMR研究）重组膜联蛋白V在大肠杆菌中产生的。在这里，我们描述了，据我们所知，第一个生物合成的高水平取代蛋氨酸2-氨基己酸（正亮氨酸），乙硫氨酸和碲蛋氨酸的蛋白质。通过电喷雾质谱、氨基酸分析和X-射线结构分析证实了这种取代。表达的条件进行了优化的频率出现的回复突变体，高水平的替代和最大的蛋白质产量。对于正亮氨酸和乙硫氨酸的掺入，E.将蛋氨酸营养缺陷型的大肠杆菌B834（DE 3）（hsd metB）在蛋氨酸限制条件下培养，在培养基中加入过量的类似物，在蛋氨酸供应耗尽后，诱导T7启动子控制下的蛋白质表达。限制碲甲硫氨酸高水平掺入蛋白质的因素是其对氧化的敏感性。为了克服这个问题，细菌生长与有限量的甲硫氨酸，收获后，其耗尽和重悬在新鲜的培养基中没有甲硫氨酸，碲甲硫氨酸被添加和蛋白质合成诱导。在这些条件下，在碲甲硫氨酸完全降解之前（数小时内），可以表达大量的蛋白质。生物合成掺入重原子如碲到重组蛋白质中可以加速获得适合于X射线结构分析的重原子衍生物的过程，补充传统的试错制备重原子衍生物用于多个同晶置换的方法。此外，氨基酸类似物的成功高水平掺入可以为详细研究蛋白质的结构和功能提供单原子突变。
We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins.