Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Rocco, C. R.; Dennison, Kirsten L.; Klenchin, Vadim A.; Rayment, Ivan; Escalante‐Semerena, Jorge C.

doi:10.1016/j.plasmid.2008.01.001

Cited by 118 publications

(121 citation statements)

References 19 publications

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“…Synaptobrevin-2 and syntaxin-1A. Full-length, cysteine-free rat synaptobrevin-2 (VAMP2) and rat syntaxin-1A were expressed separately in Escherichia coli with a N-terminal, tobacco etch virus (TEV) protease-cleavable, hexa-histidine tag from plasmid pTEV5 (59). Proteins were expressed overnight at 25°C in autoinducing media (60) The decay times were normalized to control.…”

Section: Methodsmentioning

confidence: 99%

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca 2+ -triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using singleparticle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca 2+ -independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

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Section: Methodsmentioning

confidence: 99%

“…SNAP-25. Cysteine-free SNAP-25A (C84S, C85S, C90S, and C92S) was expressed from plasmid pTEV5 (59) with an N-terminal TEV protease cleavable hexahistidine tag. The proteins were expressed overnight at 25°C in autoinducing media (60) in E. coli strain BL21 (DE3).…”

Section: Methodsmentioning

confidence: 99%

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…An overexpression vector for Mj-DFTR, pUL206, was constructed by amplifying the respective open reading frame (locus tag number MJ1536) from M. jannaschii chromosomal DNA and cloning it into the NdeI and BamHI sites of pTev5, a T7-based expression vector (77). It was designed to generate the recombinant protein with an NH 2 -terminal His 6 tag (MSYYHHHHHHDYDIPTSENLYFQ-GASH).…”

Section: Methodsmentioning

confidence: 99%

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

Susanti¹,

Loganathan²,

Mukhopadhyay

2016

Journal of Biological Chemistry

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A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavincontaining NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F 420 (F 420 H 2 ), a stronger reductant with a mid-point redox potential (E 0 ) of ؊360 mV; E 0 of NAD(P)H is ؊320 mV. Because F 420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F 420 H 2 to thioredoxin via proteinbound flavin; K m values for thioredoxin and F 420 H 2 were 6.3 and 28.6 M, respectively. The E 0 of DFTR-bound flavin was approximately ؊389 mV, making electron transfer from NAD(P)H or F 420 H 2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F 420 /F 420 H 2 pair could be as low as ؊425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F 420 -dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F 420 systems involved in microbial pathogenesis and antibiotic production.

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“…The proteins bound to the Ni-NTA resin were eluted using a step gradient of buffer A containing 20, 40, 60, 100, or 250 mM imidazole. Fractions containing MbtH were pooled and dialyzed overnight in buffer B (50 mM TrisHCl [pH 8.0], 50 mM NaCl, 10% [vol/vol] glycerol) containing 0.3 mg of hexahistidine-tagged TEV protease (34). The dialyzed and TEVdigested protein was passed through 1 ml of Ni-NTA resin to remove hexahistidine-tagged MbtH and TEV protease.…”

Section: Methodsmentioning

confidence: 99%

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

2012

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The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N 6 acylation of the central l -Lys residue and subsequent N 6 -hydroxylation of the central N 6 -acyl- l -Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N 6 -acyl- N 6 -hydroxy- l -Lys and N 6 -hydroxy- l -Lys, respectively. Furthermore, the preference of the A domain of MbtB for l -Thr over l -Ser suggests that the megasynthase produces MBT derivatives with β-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this β-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.

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Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Cited by 118 publications

References 19 publications

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

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