Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

<p>Abstract</p> <p>Background</p> <p>The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as <it>E. coli </it>due to the presence of multiple pathways for their reduction.</p> <p>Results</p> <p>Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of <it>E. coli </it>even without the disruption of genes involved in disulfide bond reduction, for example <it>trxB </it>and/or <it>gor</it>. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an <it>E. coli </it>strain with the reducing pathways intact, than in the commercial Δ<it>gor </it>Δ<it>trxB </it>strain rosetta-gami upon co-expression of Erv1p.</p> <p>Conclusions</p> <p>Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of <it>E. coli </it>and open up new possibilities for the use of <it>E. coli </it>as a microbial cell factory.</p

Sinteza i potencijalno biološko djelovanje novih 3-((N-supstituiranih indol-3-il)metilenamino)-6-amino-4-aril-pirano(2,3-c)pirazol-5-karbonitrila i 3,6-diamino-4-(N-supstituiranih indol-3-il)pirano(2,3-c)pirazol-5-karbonitrila

Starting from N-substituted indole-3-carboxaldehydes (1a-g) a series of new 3-(N-substituted indol-3-yl)methyleneamino-6-amino-4-aryl-pyrano(2,3-c) pyrazole-5-carbonitriles (3a-g and 4a-g) have been synthesized via the acid catalyzed condensation reaction of 1a-g with 3-amino-5-pyrazolone, followed by the reaction with arylidene malononitriles. A series of new 3,6-diamino-4-(N-substituted indol-3-yl)pyrano(2,3-c)pyrazole-5-carbonitriles (7a-g) have been prepared either via the base catalyzed condensation reaction of 1a-g with 3-amino-5-pyrazolone to give 6a-g, followed by the reaction with malononitrile or by the reaction of N-substituted-3-indolydine malononitriles (5a-g) with 3-amino-5-pyrazolone. According to the obtained results, the newly synthesized compounds possess significant anti-inflammatory, analgesic and anticonvulsant activities. The anticonvulsant potency of certain tested compounds was more pronounced than both anti-inflammatory and analgesic activities. Moreover, most of the newly synthesized compounds possess potential antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa.U radu se opisuje sinteza novih 3-(N-supstituiranih indol-3-il)metilenamino-6-amino-4-aril-pirano(2,3-c) pirazol-5-karbonitrila (3a-g i 4a-g) kiselo-kataliziranom kondenzacijom N-supstituiranih indol-3-karboksaldehida (1a-g) s 3-amino-5-pirazolonom iza koje slijedi reakcija s ariliden malononitrilom. Serija novih 3,6-diamino-4-(N-supstituiranih indol-3-il)pirano(2,3-c)pirazol-5-karbonitrila (7a-g) sintetizirana je reakcijom malononitrila s produktima 6a-g, koji su pripravljeni bazno-kataliziranom kondenzacijom 1a-g s 3-amino-5-pirazolonom. Neki spojevi iz serije 7a-g dobiveni su reakcijom N-supstituiranih 3-indolidin malononitrila (5a-g) s 3-amino-5-pirazolonom. Novosintetizirani spojevi imaju značajno protuupalno, analgetsko i antikonvulzivno djelovanje. Antikonvulzivno djelovanje pojedinih testiranih spojeva bilo je jače izraženo nego protuupalno i analgetsko djelovanje. Većina ispitivanih spojeva pokazuje antimikrobno djelovanje na Escherichia coli i Pseudomonas aeruginosa