Rucker P, Torti F M, Torti S V
Department of Cancer Biology, Bowman Gray School of Medicine and the Comprehensive Cancer Center of Wake Forest University, Winston-Salem, NC 27157, USA.
Protein Eng. 1997 Aug;10(8):967-73. doi: 10.1093/protein/10.8.967.
We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.
我们描述了一种创建重组铁蛋白异聚物的策略，该策略可模拟这种蛋白质的天然异质性。此方法需要使用双顺反子载体（其中两个cDNA均从该载体表达）或双载体表达策略（其中每个亚基在单个细菌宿主中从单独的兼容质粒表达），在单个细菌中共表达铁蛋白H亚基和铁蛋白L亚基的cDNA。电子显微镜和蔗糖密度梯度离心表明，铁蛋白在这类细菌中自发组装，形成预期大小和形状的具有催化活性的蛋白质。等电聚焦显示，从这些细菌中分离出的蛋白质在亚基组成上表现出有限的异质性。这种在细菌中自发组装的多亚基重组铁蛋白可能有助于铁蛋白组装和功能的进一步研究。我们的结果进一步表明，改变表达水平是改变多亚基重组蛋白中各个组分水平的一种简单方法，并且这种方法在评估各个组分对多亚基蛋白或蛋白复合物功能的贡献方面可能具有普遍用途。
