structural characterization of iron uptake from human transferrin

2010年完成了以下工作： 细菌和人蛋白的表达、纯化和络合 建立了TbpA的表达系统，可生产0.2-0.4 mg/L的纯化蛋白。用含有TbpA基因和N-末端组氨酸标签和裂解位点的质粒转化大肠杆菌的大规模制剂(27L)，用低水平的IPTG诱导过夜，然后收获。我们最近通过定向突变提高了表达产量。我们还引入了额外的蛋氨酸残基来定相TbpA晶体。提纯的TbpA在96个正在优化的孔板上形成晶体。 还建立了TbpB的表达系统，可生产7-8 mg/L的纯化蛋白。将含有TbpB基因(缺乏N端膜锚定的可溶构建体)以及N端组蛋白标签和裂解位点的质粒转化到中等规模的大肠杆菌(4L)中，用高水平的IPTG诱导几小时，然后收获。然后细胞被裂解，膜通过超速离心法分离，可溶部分在镍-NTA柱上运行。然后，镍洗脱液被进一步纯化，并可与商业上可获得的纯化的铁结合的人血清转铁蛋白结合。最近，我们首次在体外制备了TbpA-TbpB与人转铁蛋白(HTF)的结合物，并用单粒子电子显微镜对其进行了表征。我们还结晶了TbpA与HTF的络合物，并利用2007年我们求解的HTF坐标得到了晶体结构的部分分子置换溶液。 我们最近获得了TbpA+HTF(共180 kDa)的晶体，它能使X射线衍射到2.6分辨率。2010年8月，通过使用我们的HTF坐标(Wally等人，JBC 2007)和各种TonB齿状转运体模型的分子置换解决了该结构。这种结构是外膜蛋白与其全长宿主靶标(例证宿主病原体相互作用)形成复合体的第一个结构，它应该告诉我们许多关于人转铁蛋白的特异性，这种相互作用涉及的残基，可变和保守的表位位于细胞外表面(对疫苗设计有用)，如何从HTF中提取铁，以及它是如何通过外膜运输的。这一结构是一项里程碑式的成就，耗时12年完成，其重要性将在未来几个月随着我们分析数据和进行一些功能分析而显现。我们计划在2010年底之前提交一份手稿。

The following work was accomplished in 2010: Expression, Purification and Complexation of Bacterial and Human Proteins An expression system for TbpA has been developed and is capable of producing 0.2-0.4 mg/L of purified protein. A large scale preparation (27L) of E. coli transformed with a plasmid containing the gene for TbpA as well as an N-terminal His-Tag and a cleavage site are grown and induced with low level IPTG overnight and then harvested. We recently improved expression yields through targeted mutagenesis. We also introduced additional methionine residues for phasing of TbpA crystals. Purified TbpA forms crystals in 96 well plates which are being optimized. An expression system for TbpB has also been developed and is capable of producing 7-8 mg/L of purified protein. A medium scale preparation (4L) of E. coli transformed with a plasmid containing the gene for TbpB (a soluble construct lacking the N-terminal membrane anchor) as well as an N-terminal His-Tag and a cleavage site are grown and induced with high level IPTG for a few hours and then harvested. The cells are then lysed and membranes are separated via ultracentrifugation, and the soluble fraction is run over a Nickel-NTA column. The Nickel eluate is then further purified and can be bound to purified commercially available iron-bound human serum transferrin. Recently, we made the first in vitro complex of TbpA-TbpB bound to human transferrin (hTf) and this triple complex will be characterized by single particle electron microscopy. We also have crystallized TbpA in complex with hTf and have obtained a partial molecular replacement solution for the crystal structure using the coordinates of hTf which we solved in 2007. We recently obtained crystals of TbpA+hTf (180 kDa total) that diffract X-rays to 2.6 resolution. The structure was solved in August 2010 by molecular replacement using our hTf coordinates (Wally et al., JBC 2007) and various TonB-denepdent transporter models. This structure, the first of an outer membrane protein in complex with its full-length host target (exemplifying host pathogen interactions), should tell us much about the specificity for human transferrin, the residues involved in this interaction, where variable and conserved epitopes are located on the extracellular surface (useful for vaccine design), how iron is extracted from hTf, and how it is transported across the outer membrane. This structure is a landmark achievement, taking 12 years to complete, and its importance will be revealed in the coming months as we analyze the data and do some functional assays. We plan to submit a manuscript by the end of 2010.