Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin
Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin
复制标题
2[4Fe4S]-和[2Fe2S]-型梭菌铁氧还蛋白和[2Fe2S]-型农杆菌铁氧还蛋白的异源过量生产
DOI：
10.1016/j.pep.2015.12.019
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发表时间：
2016-05-01
影响因子：
1.6
通讯作者：
Wang, Shuning
中科院分区：
文献类型：
--
作者：
Huang, Haiyan;Hu, Liejie;Wang, Shuning
铁氧化还原蛋白是一类含有铁硫簇的小型酸性蛋白质，广泛存在于生物体中。它们在包括呼吸作用、光合作用、发酵、固氮、二氧化碳固定和产氢等各种代谢过程中作为电子载体发挥关键作用。然而，目前仅有几种铁氧化还原蛋白可商业化获取，这极大地限制了对铁氧化还原蛋白相关酶及代谢过程的研究。在此，我们阐述了2[4Fe4S]型和[2Fe2S]型梭菌铁氧化还原蛋白以及[2Fe2S]型农杆菌铁氧化还原蛋白的异源过量表达。向培养基中添加额外的铁源和硫源，同时使用携带pCodonplus和pRKISC质粒的大肠杆菌C41(DE3)作为宿主，极大地促进了这三种铁氧化还原蛋白中铁硫簇的合成。在 terrific 肉汤中诱导12小时后，通过亲和层析和阴离子交换层析进行纯化，从1升培养物中可获得约3.4毫克链霉亲和素（Strep）标记的和3.7毫克多聚组氨酸（His）标记的梭菌2[4Fe4S]铁氧化还原蛋白。令人兴奋的是，在 terrific 肉汤中诱导24小时后，从1升培养物中可纯化出约40毫克His标记的梭菌[2Fe2S]铁氧化还原蛋白和23毫克His标记的农杆菌[2Fe2S]铁氧化还原蛋白。重组铁氧化还原蛋白在性质和生理功能上与天然铁氧化还原蛋白明显相同。未发现两种不同亲和标签对铁氧化还原蛋白活性有负面影响。总之，我们成功开发了一种便捷的方法，能够以令人满意的产量和活性异源过量表达这三种铁氧化还原蛋白，这将对铁氧化还原蛋白相关研究非常有帮助。（C）2016爱思唯尔公司。保留所有权利。
Ferredoxins are small, acidic proteins containing iron sulfur clusters that are widespread in living organisms. They play key roles as electron carriers in various metabolic processes, including respiration, photosynthesis, fermentation, nitrogen fixation, carbon dioxide fixation, and hydrogen production. However, only several kinds of ferredoxins are commercially available now, greatly limiting the investigation of ferredoxin-related enzymes and metabolic processes. Here we describe the heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin. Adding extra iron and sulfur sources to the medium in combination with using Escherichia coli C41(DE3) harboring pCodonplus and pRKISC plasmids as host greatly enhanced iron sulfur cluster synthesis in the three ferredoxins. After induction for 12 h in terrific broth and purification by affinity chromatography and anion exchange chromatography, approximately 3.4 mg of streptavidin (Strep)tagged and 3.7 mg of polyhistidine (His)-tagged clostridial 2[4Fe4S] ferredoxins were obtained from 11 of culture. Excitingly, after induction for 24 h in terrific broth, around 40 mg of His-tagged clostridial [2Fe2S] and 23 mg of His-tagged agrobacterial [2Fe2S] ferredoxins were purified from 11 of culture. The recombinant ferredoxins apparently exhibited identical properties and physiological function to native ferredoxins. No negative impact of two different affinity tags on ferredoxin activity was found. In conclusion, we successfully developed a convenient method for heterologous overproduction of the three kinds of ferredoxins with satisfactory yields and activities, which would be very helpful for the ferredoxin-related researches. (C) 2016 Elsevier Inc. All rights reserved.