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B Rutz

Publications and source records attributed to B Rutz.

7 recordsLinked to original sources
PubMed2001

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.

Bacterial Proteins↗
PubMed2000

A dual role for BBP/ScSF1 in nuclear pre-mRNA retention and splicing.

The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion. We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and analyzed their phenotypes. While all mutants were blocked in the formation of commitment complex 2 (CC2) at non-permissive and permissive temperature, the ts mutants showed no defect in spliceosome formation and splicing in vitro. The cs mutant was defective in (pre)-spliceosome formation, but residual splicing activity could be detected. In vivo splicing of reporters carrying introns weakened by mutations in the 5' splice site and/or in the branchpoint region was affected in all mutants. Pre-mRNA leakage to the cytoplasm was strongly increased (up to 40-fold) in the mutants. A combination of ts mutants with a disruption of upf1, a gene involved in nonsense-mediated decay, resulted in a specific synthetic growth phenotype, suggesting that the essential function of SF1 in yeast could be related to the retention of pre-mRNA in the nucleus.

Adenosine Triphosphate↗
PubMed1999

Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.

DNA-Binding Proteins↗
PubMed1999

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.

Affinity Labels↗
PubMed1998

New constructs and strategies for efficient PCR-based gene manipulations in yeast.

Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphylococcus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain.

Aldose-Ketose Isomerases↗