ABSTRACT
Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.
Subject(s)
DNA, Complementary/chemistry , Fluorescent Dyes , Protein Biosynthesis , Puromycin/analogs & derivatives , Carbocyanines/chemistry , Cell-Free System/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Fluorescent Dyes/chemistry , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Proteomics/methods , Saccharomyces cerevisiae Proteins/metabolismSubject(s)
Escherichia coli Proteins/metabolism , Protein Array Analysis/methods , Proteomics/methods , Base Sequence , DNA Primers , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Fluorescent Dyes , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/isolation & purification , Green Fluorescent Proteins/metabolism , Protein Binding , Protein Interaction Mapping/methods , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
We describe the use of a DNA display system for in vitro selection of peptide ligands from a large library of peptides displayed on their encoding DNAs. The method permits completely in vitro construction of a DNA-tagged peptide library by using a wheat germ in vitro transcription/translation system compartmentalized in water-in-oil emulsions. Starting with a library of 10(9)-10(10) random decapeptides, 21 different peptide ligands were isolated for monoclonal antibody anti-FLAG M2. DNA display selected more diverse peptides with a DYKXXD consensus motif than previously reported phage display systems. Binding and recovery rates of three peptides were significantly higher than those of the original FLAG peptide, implying that these peptides would be superior to the FLAG peptide for purification of tagged proteins. The simplicity of DNA display enables two selection rounds per day to be conducted. Further, DNA display can overcome the limitations of previous display technologies by avoiding the use of bacterial cells and RNA tags. Thus, DNA display is expected to be useful for rapid screening of a wide variety of peptide ligands for corresponding receptors.
Subject(s)
DNA/analysis , Peptide Library , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Biotinylation , Cloning, Molecular , DNA/metabolism , Ligands , Oligonucleotide Array Sequence Analysis , Oligopeptides , Peptides/chemistry , Peptides/genetics , Peptides/immunology , Peptides/metabolism , Protein Biosynthesis , Streptavidin/chemistry , Transcription, GeneticABSTRACT
Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.
Subject(s)
Microscopy, Fluorescence/methods , Protein Array Analysis/methods , Animals , Cell-Free System , Cloning, Molecular , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Fluorescent Dyes/chemistry , Glass , Glutathione/chemistry , Glutathione Transferase/metabolism , HeLa Cells , Humans , Mice , Models, Chemical , Protein Binding , Protein Structure, Tertiary , Puromycin/chemistry , Puromycin/pharmacology , Recombinant Fusion Proteins/chemistryABSTRACT
We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.
