Protein encapsulation within the internal cavity of a bacterioferritin.
Protein encapsulation within the internal cavity of a bacterioferritin.
复制标题
作者：
24聚体铁蛋白的热稳定性和化学稳定性导致人们试图利用其天然存在的纳米级（8 nm）内腔用于生物技术应用。越来越感兴趣的领域是用于医学或生物催化应用的分子的封装。包封需要铁蛋白解离，通常使用高温或酸性条件（pH ≥ 2）诱导，这通常排除了脆性货物如蛋白质或肽片段的包含。在这里，我们证明了最小化盐浓度并将pH值调节至≤8.5（即低质子/金属离子浓度）可逆地改变了大肠杆菌细菌铁蛋白（Bfr）的二聚体和24聚体组装体之间的天然平衡，有利于分解形式。在这些条件下，Bfr的不同寡聚体形式之间的相互转化足够慢，以允许使用尺寸排阻色谱法获得纯二聚体和24聚体形式的野生型蛋白。这种对缔合状态的控制被利用来在组装蛋白质中不可接近的天然位点处结合血红素。生物技术应用的潜力被证明是由一个小的，酸性的[3Fe-4S]簇内的BFR内腔铁氧还蛋白的封装。每个笼捕获104 -6个带负电荷的铁氧还蛋白分子表明与内部蛋白表面的电荷互补性不是成功包封的重要决定因素。细菌铁蛋白的受控的可逆解离允许在内部空腔内捕获客体分子如蛋白质。
The thermal and chemical stability of 24mer ferritins has led to attempts to exploit their naturally occurring nanoscale (8 nm) internal cavities for biotechnological applications. An area of increasing interest is the encapsulation of molecules either for medical or biocatalysis applications. Encapsulation requires ferritin dissociation, typically induced using high temperature or acidic conditions (pH ≥ 2), which generally precludes the inclusion of fragile cargo such as proteins or peptide fragments. Here we demonstrate that minimizing salt concentration combined with adjusting the pH to ≤8.5 (i.e. low proton/metal ion concentration) reversibly shifts the naturally occurring equilibrium between dimeric and 24meric assemblies of Escherichia coli bacterioferritin (Bfr) in favour of the disassembled form. Interconversion between the different oligomeric forms of Bfr is sufficiently slow under these conditions to allow the use of size exclusion chromatography to obtain wild type protein in the purely dimeric and 24meric forms. This control over association state was exploited to bind heme at natural sites that are not accessible in the assembled protein. The potential for biotechnological applications was demonstrated by the encapsulation of a small, acidic [3Fe-4S] cluster-containing ferredoxin within the Bfr internal cavity. The capture of ∼4–6 negatively charged ferredoxin molecules per cage indicates that charge complementarity with the inner protein surface is not an essential determinant of successful encapsulation. The controlled, reversible dissociation of bacterioferritin allows the trapping of guest molecules such as proteins within the internal cavity.
登录
查看更多内容
影响因子：
3.9
作者：
通讯作者：
--
影响因子：
8
作者：
Kilic, MA;Spiro, S;Moore, GR
通讯作者：
Moore, GR
用于靶向酶定位的工程蛋白质纳米室。
影响因子：
3.7
作者：
Choudhary S;Quin MB;Sanders MA;Johnson ET;Schmidt-Dannert C
通讯作者：
Schmidt-Dannert C
影响因子：
10.8
作者：
Chakraborti, Soumyananda;Korpi, Antti;Heddle, Jonathan G.
通讯作者：
Heddle, Jonathan G.
影响因子：
14.8
作者：
Huard, Dustin J. E.;Kane, Kathleen M.;Tezcan, F. Akif
通讯作者：
Tezcan, F. Akif