title: High-throughput protein expression using a combination of ligation-independent cloning (LIC) and infrared fluorescent protein (IFP) detection.
abstract: Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here.
authors.affiliation: Institute of Biochemistry and Biology, University of Potsdam, Potsdam-Golm, Germany.
authors.forename: Hakan
authors.initials: H
authors.lastname: Dortay
authors.forename: Usha Madhuri
authors.initials: UM
authors.lastname: Akula
authors.forename: Christin
authors.initials: C
authors.lastname: Westphal
authors.forename: Marie
authors.initials: M
authors.lastname: Sittig
authors.forename: Bernd
authors.initials: B
authors.lastname: Mueller-Roeber
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D007259:Infrared Rays; D007891:Leishmania / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010843:Pichia / Q000378:metabolism; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases; D010450:Endopeptidases; C099488:TEV protease