Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.
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PMID:The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli. 222 22

Amino terminally deleted and point-mutated histidyl-tRNA synthetases were purified from E. coli via betaGal fusion proteins. A hinge region proximal and distal to the factor Xa cleavage region was necessary to cut the betaGal-fusion proteins efficiently under very mild nondenaturing conditions. N-terminal addition of either methionine or valine to this enzyme (its starting N-formyl-methionine is in vivo post-translationally removed) or the deletion of 6 amino terminal amino acids decreased the specific aminoacylation activity 2- to 7-fold. Further N-terminal deletions of 10 or 17 amino acids caused significantly reduced aminoacylation (100-fold) and ATP/PPi exchange (10-fold) activities, and a reduced binding affinity for histidine. Removal of 18 or more amino acids from the N-terminus thereby removing residues from MOTIF 1 resulted in inactive histidyl-tRNA synthetase mutants. Two point mutations within the histidyl-adenylate binding pocket, R259Q and R259K, also blocked histidyl-tRNA synthetase activity without affecting histidine or ATP binding. The experiments shown identify a highly conserved N-terminal R/KG-patch in front of MOTIF 1 as well as R259 as vital for full enzymatic activity.
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PMID:Isolation and analysis of mutated histidyl-tRNA synthetases from Escherichia coli. 926 56