内含肽的金属离子调控机理及其介导的新型哺乳动物细胞表达系统应用

内含肽（Intein）具有将自身从前体蛋白中剪切，同时将N端和C端肽链接在一起的功能，在蛋白质工程领域具有不可估量的前景。但目前人们仅能使用有限的手段如温度、pH等对Intein的活性达到较宽泛的调控，这极大限制了Intein的应用。我们前期对Intein的三维结构进行分析，提出Intein具有引入新的金属离子调控结合区域的可能，并初步得到了具有Zn离子敏感性的突变株。本项目中我们将：继续完善对金属离子调控机理的研究，提高Intein在胞内的Zn离子调控敏感性；将Zn离子研究的原理和策略拓展至其他生理必需的金属离子如Ca、Mg等，得到受其调控的新型Intein；通过对Intein结构的研究，运用新的其他调控机制的蛋白取代现有Intein的Loop区域，获得新的嵌合型Intein。本研究还首次建立Intein介导的哺乳动物细胞表达系统，为Intein介导表达的药物研究提供理论基础和实验依据。

Inteins are self-splicing polypeptides that interrupt a variety of host proteins, dividing them into two segments referred to as the N-extein and the C-extein. The splicing function performed by inteins is self-catalyzed and post-translational, and results in the excision of the intein and the ligation of the two exteins to form a mature and active host protein. Owing to this unusual ability to specifically catalyze the cleavage and formation of backbone amide bonds in proteins, intein chemistry has been exploited to develop useful applications in biotechnology. The understanding intein structure and self-splicing activity regulated through metal ion zinc is highly desirable for controlling intein function and ultimately application of the molecules. In this proposal, there are three parts the investigation are planned. First part, three directional structural analysis of an intein with focusing on metal ion zinc binding region will be characterized and mathematically calculated for proposing optimum amino acids or truncation mutagenesis for zinc binding. Then results from calculations results will be experimentally confirmed by expression of intein with a report protein. Analysis of splicing activity will be performed through in vitro and in vivo experiments. Finally the results achieved from in vitro activity analysis will be demonstrated in expression and splicing of an intein-report protein fusion under metal ion zinc regulation. Most important part of the first part, "proof-of-concept" was successfully conducted and results have shown that regulation is improved through intein point mutation. After the first part accomplished, the same strategy will be repeated by other metal ions including calcium and magnesium that are relative abundant in a cell. A result similar to zinc or even better regulation through calcium and magnesium may be achieved. Third part, again through computer simulation a new metal biding domain from different protein will be analyzed by three-dimensional structure. Then a domain with favorable calculated domain sequence will be the candidate that is inserted into the intein protein to explore intein regulation through an introduced binding region. Due to the introduction of additional metal binding domain, the intein, as we hypothesized, will be regulated very sensitively through metal ions. These results will answer key questions of intein regulation and will further contribute to realization of the intein molecules for application in protein engineering, for expression and regulation in a mammalian cell system, and in biopharmaceutical production.