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Escherichia Coli Expression System (escherichia + coli_expression_system)
Selected AbstractsCharacterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coliFEBS JOURNAL, Issue 3 2008Takayuki K. Nemoto V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His6 -tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I. [source] Specific interaction between the classical swine fever virus NS5B protein and the viral genomeFEBS JOURNAL, Issue 19 2004Ming Xiao The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3, untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3, UTR and that the NS5B protein was also able to bind the minus-strand 3, UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3, UTR more efficiently than the plus-strand 3, UTR. Further, we observed that the plus-strand 3, UTR with deletion of CCCGG or 21 continuous nucleotides at its 3, terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3, UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3, terminal. Therefore, it is indicated that the 3, CCCGG sequence of the plus-strand 3, UTR, and the 3, CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3, 21 nucleotide terminal site of both the 3, UTRs. [source] Expression, purification, crystallization and preliminary X-ray crystallographic data from TktA, a transketolase from the lactic acid bacterium Lactobacillus salivariusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Matt Horsham The enzyme transketolase from the lactic acid bacterium Lactobacillus salivarius (subsp. salivarius UCC118) has been recombinantly expressed and purified using an Escherichia coli expression system. Purified transketolase from L. salivarius has been crystallized using the vapour-diffusion technique. The crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 75.43, c = 184.11,Å, and showed diffraction to 2.3,Å resolution. [source] Purification, crystallization and preliminary X-ray diffraction analysis of human Gadd45,ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Wenzheng Zhang Gadd45, MyD118 and CR6 (also termed Gadd45,, Gadd45, and Gadd45,, respectively) comprise a family of proteins that play important roles in negative growth control, maintenance of genomic stability, DNA repair, cell-cycle control and apoptosis. Recombinant human Gadd45, and its selenomethionine derivative were expressed in an Escherichia coli expression system and purified; they were then crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 291,K using PEG 3350 as precipitant. Using synchrotron radiation, the best diffraction data were collected to 2.3,Å resolution for native crystals at 100,K; selenomethionyl derivative data were collected to 3.3,Å resolution. All the crystals belonged to space group I213, with approximate unit-cell parameters a = b = c = 126,Å. [source] Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2aACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005Wenxin Hu Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3,kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291,K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26,Å and belong to space group P21, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5,Å, , = 95.7°. [source] | ||||||||||