Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein. 1988

P H Hirel, and F Lévêque, and P Mellot, and F Dardel, and M Panvert, and Y Mechulam, and G Fayat
Laboratoire de Biochimie, CNRS UA240, Palaiseau, France.

The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

UI MeSH Term Description Entries
D008718 Methionine-tRNA Ligase An enzyme that activates methionine with its specific transfer RNA. EC 6.1.1.10. Methionyl T RNA Synthetase,Met-tRNA Ligase,Methionyl-tRNA Synthetase,Ligase, Met-tRNA,Ligase, Methionine-tRNA,Met tRNA Ligase,Methionine tRNA Ligase,Methionyl tRNA Synthetase,Synthetase, Methionyl-tRNA
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010447 Peptide Hydrolases Hydrolases that specifically cleave the peptide bonds found in PROTEINS and PEPTIDES. Examples of sub-subclasses for this group include EXOPEPTIDASES and ENDOPEPTIDASES. Peptidase,Peptidases,Peptide Hydrolase,Protease,Proteases,Proteinase,Proteinases,Proteolytic Enzyme,Proteolytic Enzymes,Esteroproteases,Enzyme, Proteolytic,Hydrolase, Peptide
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002678 Chimera An individual that contains cell populations derived from different zygotes. Hybrids,Chimeras,Hybrid
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005696 Galactosidases A family of galactoside hydrolases that hydrolyze compounds with an O-galactosyl linkage. EC 3.2.1.-. Galactosidase
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D000604 Amino Acyl-tRNA Synthetases A subclass of enzymes that aminoacylate AMINO ACID-SPECIFIC TRANSFER RNA with their corresponding AMINO ACIDS. Amino Acyl T RNA Synthetases,Amino Acyl-tRNA Ligases,Aminoacyl Transfer RNA Synthetase,Aminoacyl-tRNA Synthetase,Transfer RNA Synthetase,tRNA Synthetase,Acyl-tRNA Ligases, Amino,Acyl-tRNA Synthetases, Amino,Amino Acyl tRNA Ligases,Amino Acyl tRNA Synthetases,Aminoacyl tRNA Synthetase,Ligases, Amino Acyl-tRNA,RNA Synthetase, Transfer,Synthetase, Aminoacyl-tRNA,Synthetase, Transfer RNA,Synthetase, tRNA,Synthetases, Amino Acyl-tRNA
D001616 beta-Galactosidase A group of enzymes that catalyzes the hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-galactosides. Deficiency of beta-Galactosidase A1 may cause GANGLIOSIDOSIS, GM1. Lactases,Dairyaid,Lactaid,Lactogest,Lactrase,beta-D-Galactosidase,beta-Galactosidase A1,beta-Galactosidase A2,beta-Galactosidase A3,beta-Galactosidases,lac Z Protein,Protein, lac Z,beta D Galactosidase,beta Galactosidase,beta Galactosidase A1,beta Galactosidase A2,beta Galactosidase A3,beta Galactosidases

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