title: Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin.
abstract: 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 1 l 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 1 l 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.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China; Institute of Basic Medicine, Shandong Academy of Medical Science, Jinan 250062, People's Republic of China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors.forename: Liejie
authors.initials: L
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
authors.forename: Huili
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Tao
authors.affiliation: Environment Research Institute, Shandong University, Jinan 250100, People's Republic of China.
authors.forename: Huijun
authors.initials: H
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China. Electronic address: shuningwang@sdu.edu.cn.
authors.forename: Shuning
authors.initials: S
authors.lastname: Wang
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D002846:Chromatography, Affinity; D003013:Clostridium / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D007501:Iron
keywords: Affinity tag; Agrobacterial ferredoxin; Clostridial ferredoxin; Iron–sulfur cluster; heterologous expression