微生物学报

Plasmids	Purpose and properties	Source
pCB301-bfr	Carrying bfr gene and its native promotor for the complementation of Bfr to ferritin-deficient mutants	[24]
pCB301-dps	Carrying dps gene and its native promotor for the complementation of Dps to ferritin-deficient mutants	[24]
pUCA-19	Plasmid pUC19 carrying an A. fabrum replicon and using lac promotor to promote gene expression for the complementation of ferritin to ferritin-deficient mutants	[27]
pBfr-161^aug	pUCA-19 carrying bfr ORF to code 161 amino acids with AUG as the start code for the complementation of Bfr	This study
pBfr-161^uug	pUCA-19 carrying bfr ORF to code 161 amino acids with UUG as the start code for the complementation of Bfr	This study
pBfr-169	pUCA-19 carrying bfr ORF to code 169 amino acids with AUG as the start code for the complementation of Bfr	This study
pHis-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pHis-bfr¹⁶¹	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pBfr¹⁶⁹-His	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pBfr¹⁶¹-His	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pHis-tse-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of 58 amino acids from pET-30 (including: 6×His-thrombin site-S-Tag-enterokinase site) for the complementation of Bfr	This study
pEgfp-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and eGFP fused to the N-terminus for the complementation of Bfr	This study
pSP94-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of hepatocellular carcinoma-targeted peptide SP94 for the complementation of Bfr	This study
pBfr-169^M60L	pUCA-19 expressing Bfr with 169 amino acids and Met₆₀ was changed to Leu	This study
pET-30	Expression vector to over-express His-tagged fusion protein in E. coli	Novagen
pET-His-bfr¹⁶⁹	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶⁹-His	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the C-terminus	This study
pET-His-bfr¹⁶¹	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶¹-His	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the C-terminus	This study
pET-His-dps	pET-30 over-expressing Dps and 6×His fused to the N-terminus	This study
pET-dps-His	pET-30 over-expressing Dps and 6×His fused to the C-terminus	This study
pET-His-bfr₁₆₉^M60L	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the N-terminus	This study
pET-bfr₁₆₉^M60L-His	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the C-terminus	This study

Plasmids	Purpose and properties	Source
pCB301-bfr	Carrying bfr gene and its native promotor for the complementation of Bfr to ferritin-deficient mutants	[24]
pCB301-dps	Carrying dps gene and its native promotor for the complementation of Dps to ferritin-deficient mutants	[24]
pUCA-19	Plasmid pUC19 carrying an A. fabrum replicon and using lac promotor to promote gene expression for the complementation of ferritin to ferritin-deficient mutants	[27]
pBfr-161^aug	pUCA-19 carrying bfr ORF to code 161 amino acids with AUG as the start code for the complementation of Bfr	This study
pBfr-161^uug	pUCA-19 carrying bfr ORF to code 161 amino acids with UUG as the start code for the complementation of Bfr	This study
pBfr-169	pUCA-19 carrying bfr ORF to code 169 amino acids with AUG as the start code for the complementation of Bfr	This study
pHis-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pHis-bfr¹⁶¹	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pBfr¹⁶⁹-His	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pBfr¹⁶¹-His	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pHis-tse-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of 58 amino acids from pET-30 (including: 6×His-thrombin site-S-Tag-enterokinase site) for the complementation of Bfr	This study
pEgfp-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and eGFP fused to the N-terminus for the complementation of Bfr	This study
pSP94-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of hepatocellular carcinoma-targeted peptide SP94 for the complementation of Bfr	This study
pBfr-169^M60L	pUCA-19 expressing Bfr with 169 amino acids and Met₆₀ was changed to Leu	This study
pET-30	Expression vector to over-express His-tagged fusion protein in E. coli	Novagen
pET-His-bfr¹⁶⁹	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶⁹-His	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the C-terminus	This study
pET-His-bfr¹⁶¹	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶¹-His	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the C-terminus	This study
pET-His-dps	pET-30 over-expressing Dps and 6×His fused to the N-terminus	This study
pET-dps-His	pET-30 over-expressing Dps and 6×His fused to the C-terminus	This study
pET-His-bfr₁₆₉^M60L	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the N-terminus	This study
pET-bfr₁₆₉^M60L-His	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the C-terminus	This study

两种根癌农杆菌储铁蛋白功能的鉴定和体外自组装

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周琴 , 高苗苗 , 潘晓玥 , 王浩 , 徐楠 , 郭敏亮 ^*

微生物学报 | 研究报告 2024,64(11): 4234-4247

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微生物学报 | 研究报告 2024, 64(11): 4234-4247

两种根癌农杆菌储铁蛋白功能的鉴定和体外自组装

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周琴, 高苗苗, 潘晓玥, 王浩, 徐楠, 郭敏亮^*

作者信息

扬州大学生物科学与技术学院, 江苏扬州 225009

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

Qin ZHOU, Miaomiao GAO, Xiaoyue PAN, Hao WANG, Nan XU, Minliang GUO^*

Affiliations

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu, China

出版时间: 2024-07-19 doi: 10.13343/j.cnki.wsxb.20240272

文章导航

摘要

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【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。

关键词

根癌农杆菌 / 储铁蛋白 / 细菌铁蛋白Bfr / 来自饥饿细胞的DNA结合蛋白Dps / 蛋白质纳米笼 / 蛋白质自组装

Abstract

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[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.

Key words

Agrobacterium fabrum / ferritin / bacterioferritin (Bfr) / DNA-binding protein from starved cells (Dps) / protein nano-cage / protein self-assembly

引用本文

周琴, 高苗苗, 潘晓玥, 王浩, 徐楠, 郭敏亮. 两种根癌农杆菌储铁蛋白功能的鉴定和体外自组装. 微生物学报, 2024 , 64 (11) : 4234 -4247 . DOI: 10.13343/j.cnki.wsxb.20240272

Qin ZHOU, Miaomiao GAO, Xiaoyue PAN, Hao WANG, Nan XU, Minliang GUO. Functional identification and in vitro self-assembly of two ferritins of Agrobacterium fabrum[J]. Acta Microbiologica Sinica, 2024 , 64 (11) : 4234 -4247 . DOI: 10.13343/j.cnki.wsxb.20240272

正文

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根癌农杆菌(Agrobacterium fabrum)是一种广泛存在于土壤中的兼性植物病原菌(facultative phytopathogen)，为革兰氏阴性菌，能够侵染大多数双子叶植物，导致冠瘿瘤疾病(crown gall tumor disease)的发生^[1]。根癌农杆菌的致病机制是：将其自身质粒上的部分DNA片段转运至宿主细胞，使宿主发生遗传转化而致病^[2-3]。由于根癌农杆菌能将外源基因转入宿主并使之发生遗传转化，因此，根癌农杆菌已改造成为一种应用最广泛的植物和真菌转基因工具菌^[4-6]。

铁是生物所需微量元素中最重要的一种元素，与细胞内的各种代谢途径和生物学过程密切相关。铁在细胞中存在的离子价态为二价(Fe²⁺)和三价(Fe³⁺)。三价铁的可溶性差，生物一般只吸收和利用二价铁^[7]。然而，当细胞吸收过量的Fe²⁺后，会引发芬顿(Fenton)反应，产生活性自由基，对细胞形成毒害^[8]。为了应对过量的Fe²⁺可能对细胞造成的毒害，而又不会使细胞内缺少可利用的Fe²⁺，生物进化出一类能够维持细胞内铁平衡的储铁蛋白(ferritin)，将可溶的二价铁转变成不溶的三价铁存储在储铁蛋白内腔，当细胞需要铁时，再将三价铁还原成二价铁，从储铁蛋白内腔释放出来^[9]。

在原核生物中存在三类不同的储铁蛋白：典型的铁蛋白(classical or typical ferritin, Ftn)、细菌铁蛋白(bacterioferritin, Bfr)和来自饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)^[10]。Bfr和Dps只存在于原核生物中。Dps为12聚体纳米笼结构，被认为起着保护DNA免受氧化性破坏的作用^[11-12]。Ftn广泛存在于原核与真核生物中，不含血红素，为24聚体纳米笼结构。与Ftn一样，Bfr也是24聚体纳米笼结构，但每个24聚体Bfr含有12个血红素。Bfr的内腔能存储大量以类似铁矿石形式存在的Fe^3+[13]。一个24聚体纳米笼结构的Bfr分子可以存储多达2 700个Fe³⁺和大量的无机磷^[14-15]。

储铁蛋白的高级结构非常保守，来自不同生物的储铁蛋白外壳均为蛋白质纳米笼结构，铁存储在笼的内腔。纳米级多亚基蛋白质笼在多个领域具有广泛的应用前景^[16-18]。这类纳米笼能够在体外组装，具有生物兼容性和可降解性，可用于药物、医学影像试剂和生物活性物质的定位递送，以及纳米颗粒疫苗的制备，也可用作纳米反应器制备半导体、各种金属及其氧化物或合金的纳米颗粒；储铁蛋白的外壳是一种珍贵的纳米级蛋白质笼结构，目前已有部分储铁蛋白在特定领域实现了初步的应用；开发出更多体外重组可控的储铁蛋白，可进一步扩大其应用场景^[13,19]。Bfr由24个相同的亚基构成，2个亚基以对称的方式形成二聚体，在对称轴的位置有一个血红素，血红素中的铁与2个亚基中Met的硫络合，将2个亚基聚合在一起。12个含有血红素的二聚体进一步聚合成近似球状的24聚体笼结构。Bfr蛋白质笼的直径约为12 nm，内腔直径约为8 nm。血红素、多肽链中的某些氨基酸和所处溶液的特性严重影响24聚体纳米笼的组装^[20]。Dps由12个相同的亚基构成，Dps蛋白质笼较小，更加接近球状，其直径约为9 nm，内腔直径约为4.5 nm；Dps的内腔大约能储存500个Fe³⁺；Dps蛋白质笼结构比Bfr稳定^[19,21]。

根癌农杆菌的基因组在2001年就分别由2个独立的实验室完成测序^[22-23]。在根癌农杆菌的基因组中无编码Ftn的基因，只注释出2个储铁蛋白编码基因：一个是Bfr的编码基因atu2771，另一个是Dps的编码基因atu2477。从完成测序到现在的20多年间，根癌农杆菌基因组的注释进行了多次修正，其中对Bfr的编码基因atu2771的注释有2种不同的起始位点。一种是以AUG为起始密码子的阅读框(AAK88486.2)，编码169个氨基酸；另一种是以少见的UUG为起始密码子的阅读框(NP_355701.1和WP_006310955.1)，编码161个氨基酸。以AUG为起始密码子的阅读框N-端多8个氨基酸。此前，我们对根癌农杆菌储铁蛋白的生理功能进行了深入研究，证明储铁蛋白的缺失显著降低根癌农杆菌胞内铁含量、减弱其抗氧化性和致病能力^[24]。为进一步解析根癌农杆菌储铁蛋白的部分生物化学特性和探讨其可能的应用潜力，本研究将在蛋白质层次对根癌农杆菌的储铁蛋白的储铁能力和体外组装进行深入研究。

1 材料与方法

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1.1 菌株和质粒

根癌农杆菌(A. fabrum)野生菌株为基因组测序用的菌株C58，Ti质粒为胭脂碱型。根癌农杆菌的Bfr缺失菌株Δbfr、Dps缺失菌株Δdps和Bfr-Dps双缺失菌株ΔbdF，由C58敲除这两种蛋白质的编码基因所得，均为全编码框无痕删除突变体^[24]。大肠杆菌(Escherichia coli) DH5α菌株用作构建质粒的宿主菌，BL21(DE3)菌株用作表达蛋白质的宿主菌。大肠杆菌用LB培养基培养。根癌农杆菌用富营养培养基MG/L (mannitol-glutamate/lysogeny broth)或全合成培养基AB蔗糖培养基培养，根据细菌质粒所携带的抗性，在培养基中添加相应的抗生素，具体配方参见文献[25-26]。全合成AB蔗糖培养基中FeSO₄的浓度提高至30 μmol/L。本研究所用的质粒见表1，质粒构建采用同源重组酶将目的片段与需要的载体质粒片段相连，重组酶连接可以避免因无合适的酶切位点需要在连接处改变个别氨基酸的问题。构建质粒时，利用PCR扩增技术获取目的片段和所需的载体质粒片段。在扩增目的DNA片段引物的5′-端引入8−10个与载体质粒片段末端同源的核苷酸，同样，在扩增载体片段引物的5′-端也引入8−10个与目的片段末端同源的核苷酸。用这样设计的引物来扩增目的片段和载体片段，得到的目的片段和载体片段的两端会分别含有16−20 bp的相互同源序列，这满足了同源重组酶进行同源重组连接的要求，目的片段和载体片段的连接处不会出现因用限制酶切载体需要替换个别核苷酸的问题。构建质粒的具体步骤按产品说明书进行。表达定点突变蛋白质的质粒用PCR方法来构建。质粒通过热激转化法转入大肠杆菌中，通过电穿孔转移法转入根癌农杆菌中^[26]。

1.2 储铁蛋白的非变性聚丙烯酰胺凝胶电泳分离和铁特异性染色

根癌农杆菌的储铁蛋白经非变性凝胶电泳分离后，用亚铁氰化钾(potassium ferrocyanide)对储铁蛋白内腔的三价铁进行特异性染色形成普鲁士蓝(prussian blue)，在凝胶上可以看到清晰的蓝色条带，其他蛋白质在凝胶上不显示任何条带。细胞的收集、裂解和电泳参见文献[28-29]，并稍作修改。具体操作如下：4 000 r/min离心5 min收集等量的待测根癌农杆菌细胞，用10 mmol/L、pH 7.4的磷酸缓冲溶液洗涤2次，将细胞沉淀悬浮在200 μL细菌裂解液(50 mmol/L Tris-HCl pH 8.0，甘油体积分数20%，20 mg/mL溶菌酶，0.2 mg/mL DNase，100 mmol/L NaCl，10 mmol/L MgCl₂，1% Triton X)中，悬浮细胞在37 ℃保温30 min，室温放置1 h，然后放入液氮中速冻再融化，反复冻融2次，再加入适量直径为1 mm玻璃珠，涡旋振荡15 min，4 ℃、12 000 r/min离心20 min，上清液即为根癌农杆菌的细胞粗提液。将40 μL粗提液与10 μL电泳样品液(1mol/L Tris-HCl pH 6.8，0.25%溴酚蓝，甘油体积分数25%)混合，得到用于电泳的样品。取20 μL样品用于非变性凝胶(2.5%浓缩胶，7.5%分离胶)电泳(4 ℃，60 V恒压8 h)，分离储铁蛋白。电泳后的凝胶用酸性亚铁氰化钾溶液[150 mmol/L HCl，25 mmol/L K₄Fe(CN)₆]染色20 min^[30]。如果颜色不够深，可用3,3′-二氨基联苯胺进一步染色^[31]。

1.3 重组根癌农杆菌储铁蛋白的异源表达、纯化及组装体分析

重组蛋白的表达和纯化采用常规的方法，将蛋白质融合成具有末端His-tag的重组蛋白，利用Ni-凝胶颗粒亲和色谱纯化重组蛋白^[26,32]。将表1中能编码各种不同重组根癌农杆菌储铁蛋白的质粒分别引入大肠杆菌(E. coli) BL21(DE3)表达，表达菌培养至合适的菌密度后，加入异丙基-β-d-硫代半乳糖苷(IPTG)诱导，根据不同重组蛋白的表达情况，适当调整诱导时间和温度，4 000 r/min离心5 min收集细胞。将收集的细胞悬浮在10 mmol/L、pH 7.4的磷酸缓冲液中，用超声波破碎细胞，离心收集上清液即为含有重组根癌农杆菌储铁蛋白的粗提液。用ProteinIos Ni-IDA Resin吸附带His-tag的重组根癌农杆菌储铁蛋白，然后，用洗脱液(150 mmol/L NaCl，200 mmol/L咪唑，50 mmol/L Tris-HCl pH 7.4)将吸附的重组根癌农杆菌储铁蛋白洗下，透析去除咪唑即得纯化的重组根癌农杆菌储铁蛋白。用SDS-聚丙烯酰胺凝胶电泳鉴定重组蛋白的纯度，非变性聚丙烯酰胺凝胶电泳分析重组根癌农杆菌储铁蛋白的体外组装。

1.4 重组根癌农杆菌细菌铁蛋白的血红素重构

测定血红素对根癌农杆菌Bfr组装的影响，参考文献[33-34]的方法。首先，将纯化的Bfr重组蛋白在65 ℃解聚，然后加入氯化血红素(终浓度12 μmol/L)，保温5−15 min后，冷却至室温并放置1 h，最后将未结合的血红素透析去除。

1.5 蛋白质浓度测定

重组蛋白的分离纯化及电泳样品的蛋白质浓度测定采用考马斯亮蓝G-250比色法，以牛血清白蛋白(BSA)为标准蛋白。

1.6 根癌农杆菌Bfr结构的预测和模拟

根癌农杆菌Bfr单体的结构由Alpha-fold网站(https://alphafold.ebi.ac.uk/)预测生成。Bfr二聚体和纳米笼的结构模型由SWISS-MODEL网站(https://swissmodel.expasy.org/)模拟生成。结构图通过软件ChimeraX绘制。

2 结果与分析

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2.1 两种根癌农杆菌储铁蛋白的表达和储铁功能鉴定

根癌农杆菌基因组中注释出编码Bfr和Dps两种储铁蛋白的编码基因。尽管先前已对缺乏Bfr和Dps基因的根癌农杆菌突变体细胞内的铁含量进行了测定，但关于这两种储铁蛋白各自具体的铁储存机制与贡献，我们仍知之甚少。本研究利用亚铁氰化钾与三价铁的专一性反应形成普鲁士蓝，用以检测这两种蛋白的储铁情况。分别将根癌农杆菌Bfr、Dps的单缺失和双缺失突变体菌株，以及在这些缺失突变体中分别引入pCB301-bfr和pCB301-dps质粒的回补菌株，在AB培养基中培养48 h，收集等量细胞，提取细胞粗提液，用非变性凝胶电泳分离后亚铁氰化钾染色，结果显示，根癌农杆菌能够表达具有储铁能力的Bfr，而Bfr的缺失突变体未出现能够被亚铁氰化钾染色的蛋白质条带，所引入的Bfr回补质粒能够完全回补Bfr缺失突变体。结果还显示，无论是野生型、Bfr单缺失突变体还是Dps回补菌株均检测不到能够被亚铁氰化钾染色的Dps (图1)。

2.2 根癌农杆菌细菌铁蛋白编码框的确定

在根癌农杆菌的基因组数据中，预测的细菌铁蛋白Bfr的编码框有两种。一种是以常见的AUG为起始密码子，编码169个氨基酸。序列比对发现，169个氨基酸的根癌农杆菌Bfr阅读框比其他细菌的Bfr在N-端多8个氨基酸(图2A)，于是，将根癌农杆菌的Bfr阅读框修改为以AUG后的第9个密码子UUG为起始密码子的另一种编码框，这种编码框编码的Bfr与其他细菌中的Bfr氨基酸序列更接近。因此，在后来的基因注释中，将根癌农杆菌Bfr基因阅读框的起始密码子修改为UUG (图2A)。

以UUG为起始密码子是非常少见的，而且多数情况下表达效率也比较低。为了确定根癌农杆菌Bfr到底是以AUG为起始密码子，还是以UUG为起始密码子，我们构建了3种不同的根癌农杆菌Bfr表达质粒：pBfr-169 (用lac启动子，AUG为起始密码子表达169个氨基酸Bfr)、pBfr-161^aug (用lac启动子，AUG为起始密码子表达161个氨基酸Bfr)和pBfr-161^uug (用lac启动子，UUG为起始密码子表达161个氨基酸Bfr)。用这3种质粒分别回补根癌农杆菌的储铁蛋白缺失突变体，然后，分别检测这3种质粒是否能使突变体重新表达出能储铁的Bfr。如图2B所示，用lac启动子，以AUG为起始密码子，能表达出具有储铁能力的161个和169个氨基酸的Bfr。根据图2B中Bfr的电泳迁移距离可以判断野生型根癌农杆菌Bfr是161个氨基酸，意味着野生型是以UUG为起始密码子。然而，当用载体质粒中的lac启动子，以UUG为起始密码子(引入pBfr-161^uug质粒)时，却不能使Bfr缺失突变体重新表达出Bfr，说明野生型bfr mRNA中的UUG上游序列可能影响以UUG为起始密码子的翻译。

2.3 末端融合对根癌农杆菌细菌铁蛋白储铁功能的影响

根据模拟的Bfr结构(图3A)，多肽链的N-端在24聚体的表面，C-端在24聚体的里表面。前面的结果(图2B)也说明无论N-端是否含8个氨基酸都不影响其储铁功能。为探讨根癌农杆菌Bfr的应用潜力，需构建其融合蛋白并大量表达该蛋白，因此设计了多种融合方式(图3B)，以测试不同的融合方式对根癌农杆菌Bfr储铁功能的影响。将能表达不同融合蛋白质的质粒分别引入根癌农杆菌的储铁蛋白缺失突变体ΔbdF，检测细胞提取液中是否有能储铁的蛋白质。如图3C所示，在根癌农杆菌Bfr的C-端和161个氨基酸的Bfr¹⁶¹ N-端直接融合6个组氨酸均会使Bfr失去储铁能力。而在169个氨基酸的Bfr¹⁶⁹ N-端融合某些肽段后，尽管这些肽段的融合会影响Bfr的储铁能力，但所测试的全部Bfr¹⁶⁹ N-端融合蛋白均具有一定的储铁能力。这些结果说明Bfr¹⁶⁹ N-端多出的8个氨基酸可能为融合肽段起了连接臂的作用，降低了融合肽段对Bfr储铁功能的影响。

2.4 根癌农杆菌两种储铁蛋白的异源表达及体外组装

用大肠杆菌异源表达根癌农杆菌储铁蛋白的组氨酸标签融合蛋白，结果显示，不管是在N-端还是C-端融合6个组氨酸，根癌农杆菌的Bfr和Dps都能在大肠杆菌中大量表达，可利用融合的组氨酸纯化得到高纯度的融合蛋白(图4A)。用非变性凝胶电泳分析这些融合蛋白发现，根癌农杆菌的Bfr与其他细菌的Bfr相似，在溶液中以单体、二聚体和24聚体的形式存在(图4B)。从图4中各条带的染色程度可以看出，不同的末端融合方式对Bfr聚合体的形成影响不同，但均不会完全阻止24聚体的形成，其中Bfr¹⁶⁹的N-端融合6个组氨酸的His-bfr¹⁶⁹重组蛋白形成的24聚体最多。值得注意的是，根癌农杆菌的Dps，无论是在N-端还是在C-端融合6个组氨酸均能形成很稳定的12聚体，在凝胶电泳的泳道中看不到其他条带(图4B)。

2.5 血红素和Met₆₀替换对根癌农杆菌细菌铁蛋白体外组装的影响

细菌铁蛋白Bfr的典型特征是，在二聚体的对称轴位置有一个血红素，并且通过与2个亚基中的甲硫氨基酸形成配位键，使二聚体更易形成。根据根癌农杆菌Bfr的氨基酸序列比对(图2A)和结构模拟(图3A)结果，第60位的Met可能是与血红素络合影响二聚体形成的关键氨基酸，因此，我们构建了Met₆₀替换成Leu的根癌农杆菌Bfr变异体Bfr^M60L。通过异源表达，纯化得到了4种169个氨基酸的Bfr¹⁶⁹重组蛋白：His-bfr¹⁶⁹ (Bfr¹⁶⁹的N末端融合6个His)、Bfr¹⁶⁹-His (Bfr¹⁶⁹的C末端融合6个His)、His-bfr₁₆₉^M60L (Bfr¹⁶⁹的Met₆₀替换成Leu，N末端融合6个His)和Bfr₁₆₉^M60L-His (Bfr¹⁶⁹的Met₆₀替换成Leu，N末端融合6个His)。用血红素对这4种重组蛋白的聚合体进行重构，将经血红素处理前后的4种蛋白质进行非变性凝胶电泳分析，结果见图5A。无论是否经血红素处理或络合血红素的Met₆₀存在与否，根癌农杆菌的Bfr均能形成一定量的24聚体。然而，血红素处理显著提升了含有Met₆₀ Bfr的24聚体的量，而对变异体Bfr^M60L的24聚体的量影响很小。尽管变异体Bfr^M60L仍能形成一部分24聚体，但形成的聚合体(包括2聚体和24聚体)明显少得多，说明Met₆₀和血红素对根癌农杆菌Bfr形成24聚体是非常重要的，但并不是必不可少的。我们进一步用变异体Bfr^M60L回补根癌农杆菌的储铁蛋白缺失突变体ΔbdF，结果显示，变异体Bfr^M60L仍保留了一定的储铁功能(图5B)。

3 讨论与结论

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相当一部分细菌中同时含有Ftn、Bfr和Dps三类储铁蛋白，而在根癌农杆菌的基因组中只有2个储铁蛋白Bfr和Dps的编码基因^[24]。本研究的结果(图1)发现，根癌农杆菌的Bfr编码基因具有生物学功能，能够表达出具有储铁功能的蛋白质，Dps的编码基因在本研究的测定条件下未观察到其能表达出具有储铁功能的蛋白质。然而，早期对根癌农杆菌Dps的研究结果显示，异源表达纯化的根癌农杆菌Dps具有吸收和释放铁的能力，但不能结合DNA^[36]。本研究结果证明，异源表达的根癌农杆菌Dps能够形成稳定的12聚体，在非变性胶电泳中看不到其他条带(图4B)。这说明根癌农杆菌的Dps编码基因能够编码出有功能的Dps蛋白，但是，在用铁特异性染色检测根癌农杆菌细胞粗提液电泳结果时却看不到能储铁的Dps (图1)。我们推测，在本研究的条件下，根癌农杆菌的Dps编码基因可能未表达。因此，根癌农杆菌Dps编码基因的表达调控机理和在细胞内的准确功能值得进一步研究。从这些结果中我们也可以判断，在根癌农杆菌中起储铁和调节细胞铁平衡功能的是Bfr，而不是Dps。即使是在Bfr缺失的情况下，Dps也不能替代Bfr为细胞存储铁。

尽管三类储铁蛋白在形成聚合体时差别较大，但是亚基的结构却非常相似，都是以四段α-螺旋结构为主体；在原核生物中区分一种储铁蛋白是Ftn还是Bfr的关键是血红素，而不是亚基的氨基酸序列和三级结构，因此，依据氨基酸序列比对来注释的细菌Ftn和Bfr编码基因经常会出现错误^[8,29,37]。本研究结果显示，血红素的处理明显促进根癌农杆菌Bfr形成24聚体，Met₆₀的替换也明显减少根癌农杆菌Bfr形成24聚体，用变异体Bfr^M60L回补缺失突变体时，尽管仍有一定的储铁能力，但储铁能力显著下降，说明根癌农杆菌Bfr行使完整功能时需要血红素，因此，根癌农杆菌基因组中注释的Bfr编码基因是正确的。失去血红素络合能力的Bfr仍然具有一定的储铁能力，说明血红素不是Bfr行使功能的决定因素，因此，从某种程度上说Ftn可能是在Bfr失去血红素络合能力之后进化出来的，两者的进化关系可能非常近，只要极个别氨基酸发生突变就可能从Bfr转变成Ftn。这也进一步解释了为什么在细菌基因组的ftn和bfr基因注释中经常会出现错误。

容易获得并能方便调控其聚合和解聚的纳米笼蛋白质聚合体具有非常广阔的应用前景，特别是在医疗领域具有极高的应用价值；目前已经将个别Ftn应用到抗癌药物和医疗影像造影剂的定点递送，以及纳米颗粒疫苗的制备^{[13,35,38-39]}。要使纳米笼结构的蛋白质聚合体实现实质性的应用至少需具备3个条件：(1) 能大量制备；(2) 能够融合其他多肽或可进行适当的化学修饰；(3) 可在相对较温和的条件下控制蛋白质聚合体的解聚和聚合。根癌农杆菌的Dps蛋白不管是在N-端还是C-端融合6个His都能形成稳定的12聚体，满足可大量表达和纯化的条件，只要进一步找到控制其解聚的方法，Dps就具备了可开发应用的条件。作为抗癌药物和医疗影像造影剂定点递送的载体，需要连接上能被靶细胞识别的配基或信号肽；作为纳米颗粒疫苗的载体，也需要在表面接上抗原。本研究的结果显示，根癌农杆菌Bfr的N-端在分别融合3种不同大小的多肽或蛋白质后，仍然能够形成24聚体并具有储铁功能，说明其纳米笼结构能够承受多肽甚至蛋白质的融合，如果能够进一步改进其纳米笼的稳定性，也可以满足实质性开发应用所需的全部条件。

基金

收起

国家自然科学基金(31870118)
国家自然科学基金(32300151)
国家自然科学基金(22278350)

参考文献

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2024年第64卷第11期

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doi: 10.13343/j.cnki.wsxb.20240272

接收时间：2024-04-28
首发时间：2026-03-21
出版时间：2024-07-19

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收稿日期：2024-04-28
录用日期：2024-07-16

基金

National Natural Science Foundation of China(31870118)

国家自然科学基金(31870118)

National Natural Science Foundation of China(32300151)

国家自然科学基金(32300151)

National Natural Science Foundation of China(22278350)

国家自然科学基金(22278350)

作者信息

扬州大学生物科学与技术学院, 江苏扬州 225009

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Plasmids	Purpose and properties	Source
pCB301-bfr	Carrying bfr gene and its native promotor for the complementation of Bfr to ferritin-deficient mutants	[24]
pCB301-dps	Carrying dps gene and its native promotor for the complementation of Dps to ferritin-deficient mutants	[24]
pUCA-19	Plasmid pUC19 carrying an A. fabrum replicon and using lac promotor to promote gene expression for the complementation of ferritin to ferritin-deficient mutants	[27]
pBfr-161^aug	pUCA-19 carrying bfr ORF to code 161 amino acids with AUG as the start code for the complementation of Bfr	This study
pBfr-161^uug	pUCA-19 carrying bfr ORF to code 161 amino acids with UUG as the start code for the complementation of Bfr	This study
pBfr-169	pUCA-19 carrying bfr ORF to code 169 amino acids with AUG as the start code for the complementation of Bfr	This study
pHis-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pHis-bfr¹⁶¹	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the N-terminus for the complementation of Bfr	This study
pBfr¹⁶⁹-His	pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pBfr¹⁶¹-His	pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the C-terminus for the complementation of Bfr	This study
pHis-tse-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of 58 amino acids from pET-30 (including: 6×His-thrombin site-S-Tag-enterokinase site) for the complementation of Bfr	This study
pEgfp-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and eGFP fused to the N-terminus for the complementation of Bfr	This study
pSP94-bfr¹⁶⁹	pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of hepatocellular carcinoma-targeted peptide SP94 for the complementation of Bfr	This study
pBfr-169^M60L	pUCA-19 expressing Bfr with 169 amino acids and Met₆₀ was changed to Leu	This study
pET-30	Expression vector to over-express His-tagged fusion protein in E. coli	Novagen
pET-His-bfr¹⁶⁹	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶⁹-His	pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the C-terminus	This study
pET-His-bfr¹⁶¹	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the N-terminus	This study
pET-bfr¹⁶¹-His	pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the C-terminus	This study
pET-His-dps	pET-30 over-expressing Dps and 6×His fused to the N-terminus	This study
pET-dps-His	pET-30 over-expressing Dps and 6×His fused to the C-terminus	This study
pET-His-bfr₁₆₉^M60L	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the N-terminus	This study
pET-bfr₁₆₉^M60L-His	pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the C-terminus	This study

Plasmids

Purpose and properties

Source

pCB301-bfr

Carrying bfr gene and its native promotor for the complementation of Bfr to ferritin-deficient mutants

[24]

pCB301-dps

Carrying dps gene and its native promotor for the complementation of Dps to ferritin-deficient mutants

[24]

pUCA-19

Plasmid pUC19 carrying an A. fabrum replicon and using lac promotor to promote gene expression for the complementation of ferritin to ferritin-deficient mutants

[27]

pBfr-161^aug

pUCA-19 carrying bfr ORF to code 161 amino acids with AUG as the start code for the complementation of Bfr

This study

pBfr-161^uug

pUCA-19 carrying bfr ORF to code 161 amino acids with UUG as the start code for the complementation of Bfr

This study

pBfr-169

pUCA-19 carrying bfr ORF to code 169 amino acids with AUG as the start code for the complementation of Bfr

This study

pHis-bfr¹⁶⁹

pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the N-terminus for the complementation of Bfr

This study

pHis-bfr¹⁶¹

pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the N-terminus for the complementation of Bfr

This study

pBfr¹⁶⁹-His

pUCA-19 expressing Bfr with 169 amino acids and 6×His fused to the C-terminus for the complementation of Bfr

This study

pBfr¹⁶¹-His

pUCA-19 expressing Bfr with 161 amino acids and 6×His fused to the C-terminus for the complementation of Bfr

This study

pHis-tse-bfr¹⁶⁹

pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of 58 amino acids from pET-30 (including: 6×His-thrombin site-S-Tag-enterokinase site) for the complementation of Bfr

This study

pEgfp-bfr¹⁶⁹

pUCA-19 expressing Bfr with 169 amino acids and eGFP fused to the N-terminus for the complementation of Bfr

This study

pSP94-bfr¹⁶⁹

pUCA-19 expressing Bfr with 169 amino acids and N-terminal fusion of hepatocellular carcinoma-targeted peptide SP94 for the complementation of Bfr

This study

pBfr-169^M60L

pUCA-19 expressing Bfr with 169 amino acids and Met₆₀ was changed to Leu

This study

pET-30

Expression vector to over-express His-tagged fusion protein in E. coli

Novagen

pET-His-bfr¹⁶⁹

pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the N-terminus

This study

pET-bfr¹⁶⁹-His

pET-30 over-expressing Bfr with 169 amino acids and 6×His fused to the C-terminus

This study

pET-His-bfr¹⁶¹

pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the N-terminus

This study

pET-bfr¹⁶¹-His

pET-30 over-expressing Bfr with 161 amino acids and 6×His fused to the C-terminus

This study

pET-His-dps

pET-30 over-expressing Dps and 6×His fused to the N-terminus

This study

pET-dps-His

pET-30 over-expressing Dps and 6×His fused to the C-terminus

This study

pET-His-bfr₁₆₉^M60L

pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the N-terminus

This study

pET-bfr₁₆₉^M60L-His

pET-30 over-expressing Bfr with 169 amino acids, in which Met₆₀ was changed to Leu and 6×His was fused to the C-terminus

This study