RB
Rhoderick E. Brown
  • Faculty, University of Minnesota Austin
研究方向
  • Biochemistry, Biophysics, Cell Biology, Molecular Biology
A Simple and Straightforward Approach for Generating Small, Stable, Homogeneous, Unilamellar 1-Palmitoyl 2-Oleoyl Phosphatidylcholine (POPC) Bilayer Vesicles
一种生成小型、稳定、均质、单层 1-棕榈酰 2-油酰磷脂酰胆碱 (POPC) 双层囊泡的简单方法
作者:Yong-Guang Gao, Le Thi My Le, Amer Alam and Rhoderick E. Brown日期:12/20/2021,浏览量:2100,Q&A: 0

Various methods have been developed to generate phosphoglyceride liposomes. Approaches resulting in homogeneous populations of unilamellar bilayer vesicles are generally preferred to mimic various cell membrane situations, as well as to optimize aqueous solute trapping efficiency using the least amount of lipid for biotechnological purposes. Most are time-consuming, often tedious, or require specialized equipment, and produce vesicles with limited shelf-life at room temperature or in cold storage. Herein, we describe a straightforward approach that avoids the preceding complications and streamlines the construction of unilamellar bilayer vesicles from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/dihexanoyl phosphatidylcholine (DHPC) bicelle mixtures at room temperature. The resulting vesicles are small (32-36 nm diameter), unilamellar, bilayer vesicles that are homogeneous, stable, and resistant to freeze-thaw alterations.


Graphic abstract:



Cryo-EM of POPC vesicles formed by dilution of 0.5 q-value POPC/DHPC bicelle mix.


Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli
可溶性磷脂酶A2αC2结构域在大肠杆菌中表达后的纯化

Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase A2α C2-domain in soluble form in E. coli and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α (Hirano et al., 2019)