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SnAvi--a new tandem tag for high-affinity protein-complex purification.

ABSTRACT: Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.

SUBMITTER: Schaffer U

PROVIDER: S-EPMC2847239 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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SnAvi--a new tandem tag for high-affinity protein-complex purification.

Schäffer Ursula U Schlosser Andreas A Müller Kristian M KM Schäfer Angelika A Katava Nenad N Baumeister Ralf R Schulze Ekkehard E

Nucleic acids research 20100104 6

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. ...[more]

PMID: 20047968

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Project description:Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A, a small, bispecific molecule with affinity for both HSA and the novel target was identified. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination.

Project description:Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Dataset Information

SnAvi--a new tandem tag for high-affinity protein-complex purification.

Publications

SnAvi--a new tandem tag for high-affinity protein-complex purification.

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