title: An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability.
abstract: Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.
authors.affiliation: National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
authors.forename: Junping
authors.initials: J
authors.lastname: Fan
authors.forename: Jie
authors.initials: J
authors.lastname: Heng
authors.forename: Shuyan
authors.initials: S
authors.lastname: Dai
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.forename: Bei
authors.initials: B
authors.lastname: Zhou
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
authors.forename: Zheng
authors.initials: Z
authors.lastname: He
authors.forename: Ya
authors.initials: Y
authors.lastname: Wang
authors.forename: Taijiao
authors.initials: T
authors.lastname: Jiang
authors.forename: Xuemei
authors.initials: X
authors.lastname: Li
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Liu
authors.forename: Xianping
authors.initials: X
authors.lastname: Wang
authors.forename: Xuejun C
authors.initials: XC
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins