Protein Purification Protocols Research Articles

Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a "landmark" of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321-366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331-369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

Book Review: Protein and Peptide Analysis by LC-MS—Experimental Strategies

Protein and Peptide Analysis by LC-MS—Experimental Strategies is a publication of the Royal Society of Chemistry Chromatography Monographs series (number 15). The book consists of 12 chapters authored by researchers at the coalface of LC-MS (liquid chromatography-mass spectrometry) applications directed towards investigations of peptides and proteins, their sequences, structures, biological interactions and functions. Chapter 1 (Lottspeich) sets the scene by introducing the concepts of top down and bottom up approaches with particular emphasis on quantitative analysis. The major isotopic labelling techniques are briefly compared and this serves as a platform for examples of experiments based on these techniques described in later chapters. Chapter 2 (Letzel) provides an excellent background to LC-MS for postgraduate students and researchers embarking on research that utilises this technique. The major types of ionisation methods are introduced and examples that highlight important considerations for designing the best analytical methods are provided. This is a particularly important chapter as it alerts those relatively new to LC-MS about the importance of the choice of ionisation mode, optimising experiments by careful choice of solvents, additives, spray conditions and instrumental parameters. Chapter 3 (Ohnuma and Fukamizo) provides a commentary on the utility of recombinant expression of proteins and peptides, and presents commonly used liquid chromatography resins and methods used in protein purification. A brief outline of the experimental procedure for purification of a protein (NtChiV) expressed in E. coli is given. While the procedure presented here does not provide a detailed commentary on the challenges that may be encountered, this section certainly provides a basis for relative newcomers to the methods to know what questions need to be asked and to understand what training may be required to achieve competency in the experimental protocols for protein expression and purification. The content of Chapters 4–12 reveals the scope of the application of LC-MS to protein/peptide research and goes well beyond applications for primary sequence determination. Liquid chromatography-mass spectrometry of intact proteins is described in Chapter 4 (Young and Garcia) and is a highlight of this book. The authors emphasise the importance of optimising experimental conditions for the particular protein under study and importantly have a good grasp of the advantages and limitations of the techniques as well as being able to present a big picture view of the research area. Chapters 5 (Cheison and Kulozik) and 6 (Hoos and Niessen) deal with different aspects of LC-MS analysis of peptides derived from proteolytic or chemical digestion of proteins. The focus of the former chapter is to describe an experimental set-up for the pH stat-controlled tryptic hydrolysis of proteins. In this way, a higher degree of reproducibility may be attained. The latter describes a general method for trypsin hydrolysis of Book Review

Abstract 9466: Recombinant Mitochondrial Transcriptional Factor A Protein Attenuates Pathological Remodeling in Cardiac Myocytes

Introduction: Mitochondrial transcriptional factor A (TFAM), a nucleoid protein of mitochondrial DNA (mtDNA), is essential not only for transcription and replication but also maintenance of mtDNA. We have previously reported that, in failing myocardium, TFAM expression and the myocardial mtDNA copy number were decreased. Moreover, in myocardial infarction, overexpression of TFAM attenuated the decrease in the mtDNA copy number, ameliorated cardiac remodeling, and markedly improved survival. Since exogenously-administrated recombinant TFAM protein has been shown to enter mitochondria in cultured cells, we examined our hypothesis that recombinant TFAM protein administration attenuates pathological remodeling in cardiac myocytes. Methods and Results: We prepared recombinant human TFAM including mitochondrial targeting signal by glutathione s-transferase fusion protein purification protocol. Recombinant TFAM was successfully recruited into mitochondria of rat neonatal cardiac myocytes, whereas TFAM without mitochondrial targeting signal was observed in nucleus. Treatment with TFAM increased the mtDNA copy number dose-dependently (100 nM: 1.8±0.1-folds), whereas it did not change morphology or population of mitochondria (electron microscopy). To elucidate the mechanism of TFAM on signaling pathways in cardiac hypertrophy, we investigated the effects of TFAM on the nuclear factor of activated T cell (NFAT) signaling, which is a major transcriptional factor regulating pathological hypertrophy and remodeling. TFAM totally abolished NFAT nuclear translocation induced by both angiotensin II (41±3% to 2±1%, p<0.01) and endothelin-1 (43±3% to 3±1%, p<0.01), suppressing respective NFAT transcriptional activity and NFAT-dependent gene expression. TFAM inhibited subsequent morphological hypertrophy of cardiac myocytes (angiotensin II: 1686±37 to 1167±20 μ m 2 , p<0.01, and endothelin-1: 1759±44 to 1206±21 μ m 2 , p<0.01). Conclusion: Recombinant TFAM increases the mtDNA copy number, and attenuates angiotensin II and endothelin-1-induced hypertrophy via inhibiting NFAT signaling. Recombinant TFAM would be an attractive, novel therapeutic strategy for cardiac hypertrophy and remodeling.

High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile β-trefoil lectin domain from the mushroom Laetiporus sulphureus

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47Å resolution and also that of the [LSL(150):(lactose)β, γ)] binary complex at 1.67Å resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150) (LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.

High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = β = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

A centrifugation-free high-throughput protein purification system using in-line microfluidization

The majority of current high-throughput protein purification protocols include rate-limiting centrifugation steps. A column and nozzle assembly was developed that can be used in-line with microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols. This large-scale protein purification protocol is easy to use and widely-applicable.

Expression, purification, and refolding of active human and mouse secreted group IIE phospholipase A 2

Secreted phospholipase A 2s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A 2 (sPLA 2-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA 2-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA 2-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA 2-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA 2-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA 2-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA 2-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA 2-IIEs.

Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography

Testing several different growth and purification conditions by means of a small-scale screening is a valuable approach for identifying optimal protocols for large preparative protein productions. The availability of new mini columns for size exclusion chromatography (SEC) enables the coupling of parallel affinity purification using magnetic beads with the aggregation analysis performed by gel-filtration. Only 35 μL of bead eluate are sufficient to run both experiments and each SEC cycle needs no more than 30 min, washing and equilibration inclusive. We have shown that SEC ideally completes the SDS–PAGE analysis since it allows estimating the amount of soluble aggregates recovered in the elution fraction after affinity purification, although larger aggregates seem to be lost in the pre-column filters. SEC is also useful to identify low molecular mass contaminants that can be underestimated as they migrate together with the front of the acrylamide gel.

Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.

Production of three anti-listerial peptides by Lactobacillus curvatus in MRS broth

Three novel bioactive peptides (BAP1–3) from Lactobacillus curvatus CWBI-B28 were isolated and purified using de Man, Rogosa and Sharp (MRS) broth by a three-step protein purification protocol including ammonium sulfate precipitation, hydrophobic C18 Sep-Pak column and reverse-phase high performance liquid chromatography (RP-HPLC). This procedure allowed the recovery of chromatographically pure antimicrobial peptides with the yields of 19%, 10% and 15% of BAP1, BAP2 and BAP3, respectively. The respective apparent molecular masses as determined by tricine-SDS-polyacrilamide gel electrophoresis were 6365, 3426 and 3496. N-terminal amino acid sequencing of the BAPs and comparison of their sequences with those of international data bases indicated that BAP1 is more likely to be a casein-derived bioactive protein produced upon hydrolysis of the tryptone present in MRS broth by Lb. curvatus CWBI-B28 during active growth. However, the identity of BAP2 and BAP3 could not be determined with certainty; yet, they would be novel bacteriocins not fitting in any of the known classes of bacteriocins. Therefore, this strain would have the ability to produce intrinsic antimicrobial substances and also release bioprotective peptides from milk-proteins upon cultivation in milk or casein-containing food products due to its proteolytic activity. Thus, Lb. curratus CWBI-B28 possesses a good potential to be used in food preservation and safety.

Fully automated protein purification

Obtaining highly purified proteins is essential to begin investigating their functional and structural properties. The steps that are typically involved in purifying proteins can include an initial capture, intermediate purification, and a final polishing step. Completing these steps can take several days and require frequent attention to ensure success. Our goal was to design automated protocols that would allow the purification of proteins with minimal operator intervention. Separate methods have been produced and tested that automate the sample loading, column washing, sample elution and peak collection steps for ion exchange, metal affinity, hydrophobic interaction, and gel filtration chromatography. These individual methods are designed to be coupled and run sequentially in any order to achieve a flexible and fully automated protein purification protocol.

Two-Dimensional Differential Gel Electrophoresis of a Cell Line Derived from a Breast Cancer Micrometastasis Revealed a Stem/Progenitor Cell Protein Profile

Dissemination of primary cancer cells to distant sites is an early event in breast cancer. These cells can invade the bone marrow, rest there, and many years later disseminated tumor cells (DTC) can grow out to form overt metastases. Epithelium specific cytokeratins are commonly used as marker proteins for sensitive detection of metastatic lesions. However, due to difficulties in the detection of DTC, the question arises if DTC necessarily have the same protein expression profile as advanced tumors. On that account, we analyzed the previously uncharacterized breast cancer DTC cell line BC-M1 by 2-D DIGE. Special protein concentration and purification protocols for 2-DE were developed which resulted in high recovery rates and increased display of alkaline proteins. A broad range reference map of metastasis relevant proteins was compiled including the cytokeratins 5, 7, 8, 17, 18, and 19 and several classes of cytoskeleton proteins involved in metastasis like ezrin, gelsolin, vinculin, or vimentin. BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5 positive and cytokeratin 8/18 negative breast cancer phenotype and expresses Her-2, which is also found in stem cells/progenitor cells of primary tumors. Supported by the detection of several other epithelium-derived proteins, the example BC-M1 indicates that the protein expression profile of DTC might be reminiscent of the expression profile of the early tumor, which differs from the advanced tumor. Hence, DTC from breast cancer patients' bone marrow expressed cytokeratin 5, which further supports our hypothesis.

Proteome-Wide Identification of Novel Binding Partners to the Oncogenic Fusion Gene Protein, NPM-ALK, using Tandem Affinity Purification and Mass Spectrometry

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

High‐throughput protein production – Lessons from scaling up from 10 to 288 recombinant proteins per week

The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for handling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.

Utilization of protein splicing for purification of the human growth hormone

Developing simple and reliable methods to purify recombinant proteins is among the most important problems of modern biotechnology. It is of particular interest to take advantage of protein splicing for this purpose. Affinity tagging of inteins allows the use of regular protocols for protein purification. Autocatalytic excision of the tagged intein yields the pure protein lacking N-terminal formylmethionine. A new purification technique was developed on the basis of protein splicing for the human growth hormone. The Mxe GyrA intein with the histidine tag or cellulose-binding domain was used as a self-removable affinity unit. The resulting two-step purification protocol makes it possible to obtain the human growth hormone having the native N terminus with minimal losses.

High Affinity Interaction between a Bivalve C-type Lectin and a Biantennary Complex-type N-Glycan Revealed by Crystallography and Microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.

Application of Proteomic Techniques to Fruits and Vegetables

Proteomics and the technology supporting this science are proving to be invaluable in elucidating the tremendous complexity of biological processes. Among proteomic techniques, two-dimensional electrophoresis (2-DE) has been applied to resolve thousands of proteins and 2-DE has been especially useful for comparative studies between paired samples or populations. Improved protein extraction and purification protocols for recalcitrant fruits and vegetables have resulted in 2-DE techniques appearing in almost all fruit and vegetable proteomic studies currently being published. Numerous proteins involving various metabolic pathways have already been reported for tomato, pepper, strawberry, grape, banana, apple and pear. Significant improvements have been made to both gel and non-gel based proteomic research platforms. Better quantitative analyses and higher throughput proteomic technologies will further improve fruit and vegetable proteomic research and will have a significant impact on future breeding and post-harvest handling technologies to improve nutritional and eating quality. Keywords: Proteomics, review, 2-dimensional electrophoresis (2-DE), mass spectrometry, quality

Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

BackgroundBiopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.ResultsHere we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.ConclusionThe recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.