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A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion(More)
Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal(More)
A variety of proteins, including glycosylasparaginase, have recently been found to activate functions by self-catalyzed peptide bond rearrangements from single-chain precursors. Here we present the 1.9 A crystal structures of glycosylasparaginase precursors that are able to autoproteolyze via an N --> O acyl shift. Several conserved residues are aligned(More)
The phoB gene, which encodes a positive control factor for a number of phosphate-regulated genes in Escherichia coli, was cloned into multicopy plasmid pBR322. A phoB-cat fusion that expressed chloramphenicol transacetylase from the phoB promoter was constructed. Studies of the expression of the phoB-cat fusion showed that the pattern of regulation of the(More)
A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated(More)
Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge. The activities of enzymes with mutations in the beta-bridge segment were studied. Mutations(More)
Glycosylasparaginase uses an autoproteolytic processing mechanism, through an N-O acyl shift, to generate a mature/active enzyme from a single-chain precursor. Structures of glycosylasparaginase precursors in complex with a glycine inhibitor have revealed the backbone in the immediate vicinity of the scissile peptide bond to be in a distorted trans(More)
Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of(More)
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