高温镍铁氢化酶的异源高效表达及其成熟机理研究

The structure of the [NiFe]-hydrogenase holoenzyme is complicated and the post-translational maturation of the enzyme requires the concerted reactions of specific accessory proteins, thus heterologous expression purification is very difficult. Moreover, the experimental requirements and the molecular manipulations of hyperthermophilic archaeon are more strictly than those of mesophilic bacteria, so the research of the [NiFe]-hydrogenases from hyperthermophilic archaeon is slow. In this project, the soluble hydrogenase I (PfSHI) derived from Pyrococcus furiosus (Pf) was heterologous overexpressed in another hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk) using Tk-E.coli shuttle vector with a polyhistidine affinity tag. Through the promoter screening and construction of different expression vectors, heterologous high-yield expression system of heterotetrameric hydrogenase PfSHI was established. Then, the maturation mechanism of recombinant PfSHI (rPfSHI) in Tk was analyzed by regulating the expression of four structural subunits of PfSHI and related accessory proteins in Tk. In addition, specific endopeptidases required for PfSHI maturation in Tk were verified by constructing deletion mutants of possible endopeptidase genes, and the mechanism of PfSHI endopeptidase was studied, to provide a theoretical basis for the maturation mechanism of [NiFe]-hydrogenases. This project used Tk expression platform for heterologous expression of PfSHI and explored the mechanism of PfSHI maturation, which provides a theoretical basis for the heterologous expression of [NiFe]-hydrogenases and its application in the fields of bio-hydrogen production and hydrogen fuel cells.

高温镍铁氢化酶的结构复杂，翻译后成熟过程涉及到多个特定辅助蛋白的参与，而且高温厌氧的实验要求及分子操作相对于常温菌难度更大，导致其异源表达十分困难。本项目以极端嗜热古菌Thermococcus kodakarensis KOD1（Tk）为表达宿主，构建Tk-E.coli穿梭表达载体，以多聚组氨酸为纯化标签，建立极端嗜热古菌Pyrococcus furiosus可溶氢化酶I（PfSHI）的异源高效表达系统。在Tk的遗传背景下，研究PfSHI的成熟与其自身结构亚基和Tk中相关辅助蛋白表达量的相关性。此外，通过构建Tk中相关肽链内切酶的缺失突变体，验证PfSHI成熟所需的特异肽链内切酶，并对其蛋白结构进行分析，探索特异肽链内切酶的作用机制。本项目借助Tk蛋白表达平台进行PfSHI的异源表达并对其成熟机理进行探究，为镍铁氢化酶的异源表达及其在生物产氢、氢燃料电池等领域的应用提供一定的理论基础。

高温镍铁氢化酶的结构复杂，翻译后成熟过程涉及到多个特定辅助蛋白的参与，而且高温厌氧的实验要求及分子操作相对于常温菌难度更大，导致其异源表达十分困难。本项目以极端嗜热古菌Thermococcus kodakarensis KOD1（Tk）为表达宿主，构建Tk-E.coli穿梭表达载体，以多聚组氨酸为纯化标签，建立极端嗜热古菌Pyrococcus furiosus可溶氢化酶I（PfSHI）的异源高效表达系统。在Tk的遗传背景下，研究PfSHI的成熟与其自身结构亚基和Tk中相关辅助蛋白表达量的相关性，并探索特异肽链内切酶的作用机制。本项目首先构建了PfSHI的表达质粒，证实PfSHI能够在Tk中进行异源表达，然后通过表达载体的优化，启动子文库筛选以及过表达不同特异肽链内切酶，成功获得能够高效表达PfSHI 的 Tk 工程菌，并能够通过一步亲合层析的方法简单高效地纯化出高纯度有活性的PfSHI，并成功将其应用于体外多酶体系中，利用氢气进行还原型辅酶的再；另外，为了解决Tk菌遗传操作中转化效率低的问题，建立了具有普适性的古菌高效转化方法，为古菌研究提供了新方法新思路；为了研究四个结构亚基的作用，进行了PfSHI截短表达，并进行了生物学性质和电化学性质表征，结果表明结构亚基β和γ对于维持PfSHI的稳定性十分重要。本项目借助Tk蛋白表达平台进行PfSHI的异源表达并对其成熟机理进行探究，为镍铁氢化酶的异源表达及其在生物产氢、氢燃料电池等领域的应用提供一定的理论基础。