title: Membrane Protein Production in E. coli for Applications in Drug Discovery.
abstract: Producing high quality purified membrane proteins for structure-based drug design and biophysical assays compatible with typical timelines in drug discovery is a significant challenge. Escherichia coli has been an expression host of the utmost importance for soluble proteins and has applications for membrane proteins as well. However, membrane protein overexpression in E. coli may lead to toxicity and low yields of functional product. Here, we review the challenges encountered with heterologous overproduction of α-helical membrane proteins in E. coli and a range of strategies to overcome them. A detailed protocol is also provided for expression and screening of membrane proteins in E. coli using a His-specific fluorescent probe and fluorescent size-exclusion chromatography.
authors.affiliation: Discovery Sciences, AstraZeneca R&D, SE-43183, Mölndal, Sweden. arjan.snijder@astrazeneca.com.
authors.forename: Harm Jan Arjan
authors.initials: HJ
authors.lastname: Snijder
authors.affiliation: Discovery Sciences, AstraZeneca R&D, SE-43183, Mölndal, Sweden.
authors.forename: Jonna
authors.initials: J
authors.lastname: Hakulinen
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D002850:Chromatography, Gel; D055808:Drug Discovery / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D006801:Humans; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D046912:Multiprotein Complexes; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins
keywords: Fluorescence size exclusion chromatography; Heterologous expression of membrane proteins