两种根癌农杆菌储铁蛋白功能的鉴定和体外自组装

Functional identification and in vitro self-assembly of two ferritins of Agrobacterium fabrum

摘要:

【目的】 验证根癌农杆菌(Agrobacterium fabrum，以前也叫Agrobacterium tumefaciens)两种储铁蛋白——饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)和细菌铁蛋白(bacterioferritin, Bfr)的编码基因atu2477和atu2771的功能。确定Bfr编码基因的开放阅读框。研究末端融合、血红素和个别关键氨基酸突变对Bfr功能和体外自组装的影响。探讨两种储铁蛋白的可能应用潜力。【方法】 通过质粒将编码储铁蛋白的基因重新引入根癌农杆菌储铁蛋白缺失突变体中，回补储铁蛋白，验证回补的储铁蛋白编码基因是否能表达出具有储铁能力的储铁蛋白。用非变性凝胶电泳分离细胞粗提液中的蛋白质，铁特异性染色的方法鉴定电泳分离的蛋白质中是否有储铁蛋白。将不同的肽或蛋白质融合到储铁蛋白的末端，通过异源过量表达和纯化储铁蛋白的重组蛋白，用非变性凝胶电泳分析这些重组蛋白在体外的自组装。用血红素重构处理和氨基酸定点突变的方法研究血红素和个别关键氨基酸对Bfr功能和体外自组装的影响。【结果】 非变性凝胶电泳和铁特异性染色结果显示，在根癌农杆菌的野生菌株、其相关突变体以及对应的回补菌株中，均仅检测到Bfr的表达，未检测到Dps的存在。当分别回补能编码161个和169个氨基酸Bfr的基因后，发现野生型菌株中的Bfr与回补编码161个氨基酸Bfr的回补菌株一样大。多肽和蛋白质的末端融合对Bfr的功能和自组装有一定影响，但不会使Bfr完全失去功能和自组装能力。结果还表明，血红素和预测可络合血红素铁的Met₆₀的替换也只影响Bfr的功能和自组装，并未使Bfr功能完全丧失。【结论】 根癌农杆菌主要通过Bfr存储铁元素。bfr基因的开放阅读框(open reading frame, ORF)以少见的UUG为起始密码子，编码产生包含161个氨基酸的蛋白质，而非169个氨基酸。根癌农杆菌的dps基因在本文的测定条件下均处于不表达状态。根癌农杆菌的Bfr和Dps蛋白均比较稳定，能够承受末端的多肽或蛋白质融合，不会使蛋白质的结构完全破坏，因此，经适当改造后具有开发应用的潜力。

Abstract:

[Objective] This study aims to validate the functions of two ferritin-encoding genes: bacterioferritin (Bfr)-encoding gene (atu2771) and DNA-binding protein from starved cells (Dps)-encoding gene (atu2477), in Agrobacterium fabrum, to determine the open reading frame (ORF) of the Bfr-encoding gene, to investigate the effects of terminal fusion, heme group, and key residues on the function and self-assembly of A. fabrum Bfr, and to explore the potential applications of the two ferritin nano-cages. [Methods] We re-introduced the two ferritin-encoding genes into the ferritin-deficient mutants of A. fabrum respectively via plasmids to verify if the re-introduction could complement the ferritins of the ferritin-deficient mutants and thus validate the functions of the two genes. Native PAGE was employed to separate the ferritins in the crude extract of A. fabrum and potassium ferrocyanide (an iron-specific staining reagent) was used to stain the ferritins. Various peptides or protein were fused to the termini of two ferritins to test if the terminal fusion would affect the functions and self-assembly of the two ferritins. Site-directed mutation was then employed to test the effects of the key residue and heme group on the function and self-assembly of Bfr. [Results] Iron-specific staining on the ferritins separated by native PAGE showed that the Bfr-encoding gene expressed Bfr in all the tested A. fabrum strains, whereas the Dps-encoding gene expressed Dps in none of the tested A. fabrum strains. Complementary experiment with two different Bfr-encoding ORFs (encoding 161 residues and 169 residues) showed that Bfr in the wild type was encoded by the ORF encoding 161 residues. The results demonstrated that terminal fusions with different peptides or protein influenced but did not abolish the function and self-assembly of Bfr. The substitution of Met₆₀, which was predicted to chelate the iron of heme, indicated that heme affected the function and self-assembly of Bfr but was not indispensable. [Conclusion] A. fabrum utilizes Bfr to store iron. The ORF of the Bfr-encoding gene utilizes uug (a rare start code) as its start code and encodes a Bfr composed of 161 residues. The Dps-encoding gene of A. fabrum expressed in none of the tested conditions. The structures of both Bfr and Dps of A. fabrum are stable enough to withstand the terminal fusion with various peptides or protein, suggesting that both Bfr and Dps nano-cages demonstrate great promise for biotechnological applications.