High-throughput, fluorescence-based screening for soluble protein expression.
Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.
Coleman
M A
MA
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Ave., L-448, Livermore, California 94550, USA.
Lao
V H
VH
Segelke
B W
BW
Beernink
P T
PT
D001896:Boron Compounds / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011145:Polyvinyls / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D005465:Fluorocarbon Polymers
D001896:Boron Compounds; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D011145:Polyvinyls; D012346:RNA, Transfer, Amino Acyl; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers