A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium.
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ABSTRACT:
SUBMITTER: Malten M
PROVIDER: S-EPMC1392972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The NiFe2O4 magnetic nanoparticles (NF-MNPs) were prepared for one-step selective affinity purification and immobilization of His-tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier-transform infrared spectrophotometer and microscopy. The immobilization and purification of His-tagged GluDH on NF-MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris-HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg-1 support, respectively. The immobilized GluDH exhibited high thermostability, pH-stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni-NTA resin, the NF-MNPs displayed a higher specific affinity with His-tagged recombinant GluDH.
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Project description:BackgroundCell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.AimTo develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.ResultsWe have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.ConclusionsThe small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.
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Project description:A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.
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Project description:Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria for physiological and biochemical analyses from plant cells is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in plant cells with a triple haemagglutinin-tag for rapid purification by a single affinity purification step. This protocol yields highly purified mitochondria from 1 g of Arabidopsis seedlings that are suitable for enzyme activity measurements and delivers sufficient amounts of mitochondrial proteins for deep proteomic profiling. We demonstrate that the method can be applied to proteomic analysis of an Arabidopsis mutant deficient in the mitochondrial glutamate transporter À bout de souffle (BOU) and we identify 27 differentially expressed proteins in the bou mutant. Our work also sets the stage for the development of advanced isolation protocols for mitochondria from distinct cell types.
2023-11-14 | PXD014137 | Pride
Project description:Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
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Project description:BackgroundDuring the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of beta-lactam antibiotics were systematically improved.ResultsFor this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached.ConclusionThe combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.
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Project description:Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.
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Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.
2009-05-12 | GSE13654 | GEO
Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species. strain_or_line_design
2010-06-25 | E-GEOD-13654 | biostudies-arrayexpress
Project description:Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His6-tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.
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