title: Rapid screening of membrane protein expression in transiently transfected insect cells.
abstract: Membrane proteins play critical roles in many biological processes and are the focus of intense biomedical research. One bottleneck for studying membrane proteins is the difficulty in expressing correctly folded and stable proteins, which often requires extensive protein engineering and multiple rounds of optimization, a time and resource intensive process. Here, we describe a method for rapidly screening membrane protein expression in insect cells. The method uses a green fluorescent protein (GFP) covalently fused to target membrane proteins and the resulting fusion proteins are then transiently expressed in insect cells. This approach enables us to dramatically reduce the time and resources required for expression screening by eliminating the need to create recombinant baculovirus. We show that transiently expressed membrane proteins can be directly monitored for their subcellular localizations by fluorescence microscopy. Moreover, their expression levels, approximate molecular mass, and stability can be evaluated with nanogram levels of unpurified proteins by ultrasensitive fluorescence-detection size exclusion chromatography (FSEC). We present our proof of principle studies using a homotrimeric ion channel (ASIC3) and a heterodimeric transporter (SLC7A5/SLC3A2) as examples, and demonstrate the utility of transient expression coupled with FSEC in optimizing membrane protein expression.
authors.affiliation: Department of Protein Technologies, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, United States. haoc@amgen.com
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Paul L
authors.initials: PL
authors.lastname: Shaffer
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.forename: Paul E
authors.initials: PE
authors.lastname: Rose
mesh_terms: D062565:Acid Sensing Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D016367:Baculoviridae; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007313:Insecta / Q000166:cytology / Q000235:genetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
chemical_list: C112231:ASIC3 protein, human; D062565:Acid Sensing Ion Channels; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; D008565:Membrane Proteins; D011994:Recombinant Proteins; C545989:SLC3A2 protein, human; D049452:Green Fluorescent Proteins