镍螯合纳米脂蛋白颗粒固定组氨酸标记蛋白。

Immobilization of His-tagged proteins on nickel-chelating nanolipoprotein particles.

机构信息

Chemistry, Materials, Earth, and Life Sciences, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.

出版信息

Bioconjug Chem. 2009 Mar 18;20(3):460-5. doi: 10.1021/bc8003155.

PMID:19239247

Abstract

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.

摘要

纳米脂蛋白颗粒（NLPs）是纳米大小的盘状颗粒，可从纯化的载脂蛋白和磷脂自组装而成。它们的大小和易于功能化表明，NLPs 作为展示和递呈重组蛋白的平台具有潜在的应用。为此，我们研究了将镍螯合脂质掺入 NLPs（NiNLPs）中，以及随后螯合多组氨酸（His）标记蛋白。从最初的用于 NLP 形成的脂质筛选中，两种磷脂 DMPC 和 DOPC 被确定为合适的主体脂质，可将镍螯合脂质 DOGS-NTA-Ni 掺入 NLPs 中，并且可以用不同量的 DOGS-NTA-Ni 成功形成 NiNLPs。由 10％DOGS-NTA-Ni 和 90％主体脂质（DMPC 或 DOPC）组成的 NiNLPs 通过尺寸排阻色谱（SEC）、非变性梯度凝胶电泳（NDGGE）和原子力显微镜（AFM）进行了全面表征。三种不同的 His 标记蛋白以镍依赖性方式被捕获在 NiNLPs 上，固定化蛋白的量取决于 NiNLP 的大小和组成。