structural characterization of iron uptake from human transferrin

人转铁蛋白吸收铁的结构特征

基本信息

批准号：
7967375
负责人：
Susan Buchanan
金额：
$ 34.43万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7967375
关键词：
Amino Acids Anions Bacteria Bacterial Infections Bacterial Meningitis Binding Cells Complex Cytolysis Disease Electron Microscopy Escherichia coli Excision Family member Genes Goals Gram-Negative Bacteria Haptoglobins Harvest Hemoglobin Hour Human In Transferrin In Vitro Individual Iron Iron-Binding Proteins Isopropyl Thiogalactoside Lactoferrin Length Liquid substance Lobe Mediating Membrane Membrane Proteins Metals Methionine Methods Molecular Mutagenesis N-terminal Neisseria meningitidis Nickel Phase Plasmids Preparation Process Proteins Role Route Running Sequence Homology Serum Site Solutions Structure System Transferrin Transferrin-Binding Protein A Transferrin-Binding Protein B Transferrin-Binding Proteins Ultracentrifugation Vaccines Work base improved particle pathogenic bacteria protein complex protein structure receptor therapeutic development uptake

项目摘要

The following work was accomplished in 2009: 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 transferring (hTf) and this 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 aim to provide structures of TbpA alone, TbpA-hTf, and TbpA-TbpB-hTf in the coming year.

2009年主要完成了以下工作：细菌和人类蛋白质的表达、纯化和复合 TbpA 表达系统已经开发出来，能够产生 0.2-0.4 mg/L 的纯化蛋白。将用含有 TbpA 基因以及 N 末端 His 标签和切割位点的质粒转化的大肠杆菌的大规模制备物 (27L) 生长并用低水平 IPTG 诱导过夜，然后收获。我们最近通过定向诱变提高了表达产量。我们还引入了额外的蛋氨酸残基用于 TbpA 晶体的定相。纯化的 TbpA 在正在优化的 96 孔板中形成晶体。 TbpB 表达系统也已开发出来，能够产生 7-8 mg/L 的纯化蛋白。使用含有 TbpB 基因（缺乏 N 端膜锚的可溶性构建体）以及 N 端 His 标签和切割位点的质粒转化的大肠杆菌的中等规模制备物 (4L) 进行生长和诱导用高浓度 IPTG 处理几个小时，然后收获。然后裂解细胞并通过超速离心分离膜，并将可溶部分过镍-NTA 柱。然后将镍洗出液进一步纯化，并可与纯化的市售铁结合人血清转铁蛋白结合。最近，我们在体外制备了第一个 TbpA-TbpB 与人转移 (hTf) 结合的复合物，该复合物将通过单粒子电子显微镜进行表征。我们还结晶了与 hTf 复合的 TbpA，并使用我们在 2007 年解决的 hTf 坐标获得了晶体结构的部分分子置换解。我们的目标是提供单独的 TbpA、TbpA-hTf 和 TbpA-TbpB- 的结构。 hTf 在来年。