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首页> 外文学位 >High Throughput Engineering Of Split Intein With Improved Trans-Splicing Kinetics And Novel Split Intein Mediated Modular Synthesis.
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High Throughput Engineering Of Split Intein With Improved Trans-Splicing Kinetics And Novel Split Intein Mediated Modular Synthesis.

机译：具有改进的反式剪接动力学和新型拆分内含蛋白介导的模块合成的拆分内含蛋白的高通量工程。

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摘要

Inteins are internal elements that can be found in organisms in all three domains of life and offer a number of attractive methods for biotechnology applications. The applications of intein include tagless protein purification, biosensors, and tools for pharmaceutical research. However, there are some drawbacks limiting the applications of intein. Intein's trans-splicing reactions can be a powerful tool on the protein engineering field, but split inteins usually have low splicing efficiency. Also, the reassembly of exteins usually involves only two domains; the assembly of more than two exteins cannot be accomplished. A focus in our lab, therefore, has been to overcome its limitations to make intein a more versatile tool and to discover new applications for intein. We first developed a reporter assay based on the subcellular localization of the reverse tetracycline-controlled transcriptional activator (rtTA) to engineer Nostoc punctiforme PCC73102 DnaE split intein into a temperature sensitive intein as well as to improve its splicing efficiency. Second, we developed a novel intein system that can be applied to splice multiple-domain proteins. Trans-splicing is used to implement novel split intein mediated modular synthesis (SIMMS) to allow the post translational splicing of more than two exteins. These newly developed strategies significantly enhance the utility of inteins in biotechnology studies.
机译：内含子是可以在生命的所有三个领域的生物中发现的内部元素，并为生物技术应用提供了许多有吸引力的方法。内含肽的应用包括无标签蛋白纯化，生物传感器和药物研究工具。但是，存在一些限制内含肽应用的缺点。内含肽的反式剪接反应可能是蛋白质工程领域的有力工具，但已分离内含肽的剪接效率通常较低。同样，重组蛋白通常仅涉及两个结构域。无法完成两个以上蛋白的组装。因此，我们实验室的重点是克服其局限性，以使内含肽成为更通用的工具，并发现内含肽的新应用。我们首先开发了一种基于反向四环素控制的转录激活子（rtTA）的亚细胞定位的报告基因检测方法，以将点状Nostoc点状PCC73102 DnaE片段内含子改造成对温度敏感的内含子，并提高其剪接效率。第二，我们开发了一种新型的intein系统，可用于剪接多域蛋白。反式剪接用于实现新型的拆分内含子介导的模块合成（SIMMS），以允许翻译后剪接两个以上的内在蛋白。这些新开发的策略显着增强了intein在生物技术研究中的实用性。

著录项

  • 作者

    Li, Naiyi.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Engineering Chemical.;Chemistry Biochemistry.
  • 学位 M.S.
  • 年度 2014
  • 页码 64 p.
  • 总页数 64
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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