Vectors for the expression of tagged proteins in Schizosaccharomyces pombe

Craven, Rachel A.; Griffiths, Dominic J. F.; Sheldrick, K S; Randall, Richard E.; Hagan, Iain; Carr, Antony

doi:10.1016/s0378-1119(98)00434-x

Cited by 211 publications

(198 citation statements)

References 29 publications

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“…Using pNMT1-TOPO  the protein of interest is expressed with two C-terminal tags, a hexahistidine tag and a Pk tag, respectively; the Pk tag represents an immunological epitope that has been shown to work very well in Sz. pombe (Bureik et al, 2002;Craven et al, 1998). Plasmid pGEM3Z-CYP21 (Nikoshkov et al, 1997), the human CYP21 cDNA, was a kind gift from Walter L. Miller (University of California, San Francisco, CA).…”

Section: Expression Vector and Cyp21 Cdnamentioning

confidence: 99%

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

Drăgan¹,

Blank²,

Bureik

2006

Yeast

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Cytochrome P450s are haem-containing monooxygenases that catalyse a variety of oxidations utilizing a large substrate spectrum and are therefore of interest for biotechnological applications. We expressed human CYP21 in fission yeast Schizosaccharomyces pombe as a eukaryotic model for P450-dependent whole-cell biotransformation. The resulting strain displayed strong steroid hydroxylase activity that was accompanied by contrary effects on respiration and non-respiratory oxygen consumption, which combined to a significant decline in total oxygen consumption of the cells. While production of ROS (reactive oxygen species) decreased, the TCA cycle activity increased, as was shown by metabolic flux (METAFoR) analysis. Pentose phosphate pathway (PPP) activity was found to be negligible, regardless of growth phase, CYP21 expression or biocatalytic activity, indicating that NADPH levels in Sz. pombe are sufficiently high to support an exogenous P450 without adaptations of central carbon metabolism. We conclude from these data that neither oxygen supply nor NADPH availability are limiting factors in P450-dependent biocatalysis in Sz. pombe.

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Section: Expression Vector and Cyp21 Cdnamentioning

confidence: 99%

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

Drăgan¹,

Blank²,

Bureik

2006

Yeast

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“…klp2 ϩ was tagged at its C terminus with three tandem repeats of the pk1 tag (Craven et al, 1998) followed in frame by green fluorescent protein (GFP; S65T allele; Heim and Tsien, 1996). The ura4 ϩ gene was placed downstream of the tags to serve as a marker for the strain.…”

Section: Construction Of the Klp2 ؉ -Pk-gfp Homologous Integrantmentioning

confidence: 99%

pkl1⁺andklp2⁺: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

Troxell

Sweezy

West

et al. 2001

MBoC

114

136

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We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2 ϩ nor pkl1 ϩ is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7 ts , a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2⌬,dhc1-d1 in karyogamy, pkl1⌬,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1⌬ shortens it. The anaphase spindle of klp2⌬ becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.

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“…The amino terminal half (the first 2302 base pairs) of the myo2 ϩ gene was isolated from the plasmid pBluemyo2 ϩ (May et al, 1997) as an SalI-BamHI fragment and was ligated into SalI-BamHI cut pREP41Negfp ϩ (Craven et al 1998) to give the plasmid pREFP41gfp-myo2 768 . The carboxyl-terminal half (the last 2275 base pairs) of the myo2 ϩ gene was isolated from pBluemyo2 ϩ as a BamHI-BamHI fragment and was ligated into the BamHI site of pREP41gfp-myo2 768, to give pREP41gfp-myo2 ϩ .…”

Section: Molecular Genetic Manipulationsmentioning

confidence: 99%

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Mulvihill

Barretto

Hyams

2001

MBoC

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Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S1518A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S1518E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25°C but not at 36°C. GFP-Myo2S1518E rings persisted at 36°C incdc7-24 but not in another SIN kinase mutant,sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy ofmyo2 + was fused to GFP (strainmyo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36°C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.

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Vectors for the expression of tagged proteins in Schizosaccharomyces pombe

Cited by 211 publications

References 29 publications

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

pkl1⁺andklp2⁺: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

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Vectors for the expression of tagged proteins in Schizosaccharomyces pombe

Cited by 211 publications

References 29 publications

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

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pkl1⁺andklp2⁺: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis