Tandem Affinity Purification of Proteins

Günzl, Arthur; Schimanski, Bernd

doi:10.1002/0471140864.ps1919s55

Cited by 28 publications

(25 citation statements)

References 20 publications

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“…Isolated mitochondria from 7.2 ϫ 10 10 cells were solubilized as previously described by resuspending (400 g of mitochondria/40 l) in solubilization buffer A (50 mM NaCl, 50 mM imidazole/HCl, 2 mM 6-aminohexanoic acid, 1 mM EDTA, pH 7.0) containing 2% dodecyl maltoside in a rotator for 30 min at 4°C (31). PTP-tagged protein complexes were subsequently purified as described previously (36). Samples from the tandem affinity purification were examined by 8 -10% polyacrylamide gel electrophoresis and immunoblot with anti-Protein C (Delta Biolabs).…”

Section: Inducible Expression Of Variant Trnamentioning

confidence: 99%

Mitochondrial Membrane Complex That Contains Proteins Necessary for tRNA Import in Trypanosoma brucei

Seidman

Johnson

Gerbasi

et al. 2012

Journal of Biological Chemistry

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Section: Inducible Expression Of Variant Trnamentioning

confidence: 99%

Mitochondrial Membrane Complex That Contains Proteins Necessary for tRNA Import in Trypanosoma brucei

Seidman

Johnson

Gerbasi

et al. 2012

Journal of Biological Chemistry

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“…Future endeavors should be directed toward the use of a combination of biochemistry, bioinformatics, genetics, and proteomics approaches for in-depth exploration of the trypanosome-specific regulators and integration of these regulators into the existing pathways. For example, tandem affinity purification of protein complexes provides an unparalleled method for identification of novel partners of known cell cycle regulators (40,65). A forward genetics approach, through an RNAi screen, can also provide great promise for identification of novel cell cycle regulators (78), but an efficient mutant selection scheme is necessary to distinguish between the true cell cycle regulators and those playing indirect roles in cell cycle control.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Regulation of the Cell Division Cycle in Trypanosoma brucei

Li¹

2012

Eukaryot Cell

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The cell division cycle is tightly regulated by the activation and inactivation of a series of proteins that control the replication and segregation of organelles to the daughter cells. During the past decade, we have witnessed significant advances in our understanding of the cell cycle in Trypanosoma brucei and how the cycle is regulated by various regulatory proteins. However, many other regulators, especially those unique to trypanosomes, remain to be identified, and we are just beginning to delineate the signaling pathways that drive the transitions through different cell cycle stages, such as the G 1 /S transition, G 2 /M transition, and mitosis-cytokinesis transition. Trypanosomes appear to employ both evolutionarily conserved and trypanosome-specific molecules to regulate the various stages of its cell cycle, including DNA replication initiation, spindle assembly, chromosome segregation, and cytokinesis initiation and completion. Strikingly, trypanosomes lack some crucial regulators that are well conserved across evolution, such as Cdc6 and Cdt1, which are involved in DNA replication licensing, the spindle motor kinesin-5, which is required for spindle assembly, the central spindlin complex, which has been implicated in cytokinesis initiation, and the actomyosin contractile ring, which is located at the cleavage furrow. Conversely, trypanosomes possess certain regulators, such as cyclins, cyclin-dependent kinases, and mitotic centromere-associated kinesins, that are greatly expanded and likely play diverse cellular functions. Overall, trypanosomes apparently have integrated unique regulators into the evolutionarily conserved pathways to compensate for the absence of those conserved molecules and, additionally, have evolved certain cell cycle regulatory pathways that are either different from its human host or distinct between its own life cycle forms.

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“…Since the advent of this technology (71), the TAP tag and the TAP method have been modified in various ways to accommodate different systems, extracts, or protein complexes (26). For the purification of nuclear protein complexes in trypanosomes, the PTP (protein C epitope-TEV protease cleavage site-protein A domains) modification of TAP has proven to be very useful (26,72). One of the first applications of the PTP tag was the purification of the trypanosome U1 snRNP.…”

Section: Tandem Affinity Purification Of Splicing Complexes In T Bruceimentioning

confidence: 99%

The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

2010

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Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei, Trypanosoma cruzi, and several species of Leishmania cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL) trans splicing in T. brucei established a key difference between parasites and hosts. In SL trans splicing, the capped 5-terminal region of the small nuclear SL RNA is fused onto the 5 end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by cis splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to ϳ170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in T. brucei, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.Trypanosomatids are protistan parasites infecting hosts as diverse as mammals, insects, and plants. In humans, vectorborne Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. cause lethal diseases, and the strong impact of these parasites on global health has spurred investigations of the molecular processes in these organisms from early on. One of the first key discoveries in regard to gene expression was spliced leader (SL) trans splicing, which was eventually found to be an essential maturation step for all nuclear pre-mRNA in trypanosomatids.The initial discoveries of SL trans splicing were made in T. brucei, and until now, this organism has remained the preferred trypanosomatid organism for spliceosomal studies. T. brucei is an extracellular parasite which evades the mammalian immune system by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG expression has therefore been a research focus, and it was on VSG mRNAs that the 5Ј-terminal region was first discovered to contain a leader sequence which was not encoded in the VSG gene (10, 87). Further analysis showed that the 39-nucleotide (nt)-long leader was derived from the 5Ј terminus of a separate, small nuclear RNA, which has been termed SL RNA or miniexon-derived RNA (12,34,56). The discovery of a Y structure intermediate which corresponds to the cis splicing intron-exo...

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Tandem Affinity Purification of Proteins

Cited by 28 publications

References 20 publications

Mitochondrial Membrane Complex That Contains Proteins Necessary for tRNA Import in Trypanosoma brucei

Mitochondrial Membrane Complex That Contains Proteins Necessary for tRNA Import in Trypanosoma brucei

Regulation of the Cell Division Cycle in Trypanosoma brucei

The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

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