ABSTRACT
A previously engineered Methanocaldococcus jannaschii tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting tRNA(CUA Tyr)-tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Archaea/enzymology , RNA, Transfer/genetics , Amino Acyl-tRNA Synthetases/genetics , Animals , Base Sequence , Blotting, Western , Cell Line , Humans , Mammals , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer/chemistryABSTRACT
The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation, and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that selectively forms a covalent linkage to the target protein. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide, TOP1, that weakly binds to the target protein, the Src homology 3 (SH3) domain of human Abelson tyrosine kinase (Abl), was fused to green fluorescent protein (GFP) and l-DOPA was site-specifically incorporated into the peptide region (TOP1-DOPA-GFP). TOP1-DOPA-GFP produced from Escherichia coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.
Subject(s)
Blotting, Western/methods , Peptides/metabolism , Proto-Oncogene Proteins c-abl/analysis , Amino Acid Sequence , Cross-Linking Reagents/metabolism , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Levodopa/genetics , Levodopa/metabolism , Peptides/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques , src Homology DomainsABSTRACT
Come together right now with L-DOPA: Chemical cross-linking is widely used to study protein-protein interactions. However, many cross-linking agents suffer from low reactivity or selectivity. An efficient and selective reaction of site-specific protein cross-linking was achieved using genetically incorporated 3,4-dihydroxy-L-phenylalanine.
Subject(s)
Codon, Terminator/genetics , Cross-Linking Reagents/chemistry , Dihydroxyphenylalanine/chemistry , Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Codon, Terminator/metabolism , Cysteine Endopeptidases/metabolism , Mutant Proteins/metabolism , Protein Engineering , RNA, Messenger/metabolism , Tyrosine-tRNA Ligase/metabolismABSTRACT
A mammalian two-hybrid system (termed as trM2H) was developed to detect protein-protein interactions in vivo, based on the reconstitution of the functions the of tetracycline repressor (TetR). The system is sensitive enough to detect protein-protein interactions with K(d) up to 55microM in mammalian cells, and the system can be regulated by small molecules. This system can be used as an efficient genetic selection system to map protein-protein interactions in mammalian cells.
