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 ce

SUBMITTER: Dortay H 

PROVIDER: S-EPMC3082538 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature