Sufficient Amounts Of Protein Research Articles

Recent advances in the production of proteins in insect and mammalian cells for structural biology

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

Expression and purification of recombinant human EGFL7 protein

The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

E. coli expression and purification of human and cynomolgus IL-15

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His) 6-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His) 6-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey ( Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3 − CD8 + lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.

Purification of Proteins Associated with Specific Genomic Loci

Eukaryotic DNA is bound and interpreted by numerous protein complexes in the context of chromatin. A description of the full set of proteins that regulate specific loci is critical to understanding regulation. Here, we describe a protocol called proteomics of isolated chromatin segments (PICh) that addresses this issue. PICh uses a specific nucleic acid probe to isolate genomic DNA with its associated proteins in sufficient quantity and purity to allow identification of the bound proteins. Purification of human telomeric chromatin using PICh identified the majority of known telomeric factors and uncovered a large number of novel associations. We compared proteins found at telomeres maintained by the alternative lengthening of telomeres (ALT) pathway to proteins bound at telomeres maintained by telomerase. We identified and validated several proteins, including orphan nuclear receptors, that specifically bind to ALT telomeres, establishing PICh as a useful tool for characterizing chromatin composition.

Studies on Chemical Composition and Nutritive Evaluation of Wild Edible Mushrooms

Agaricus bisporus and Agaricus bitorquis are among most favorite mushrooms of the world. Mineral contents, proximate composition and fatty acid profile of these mushrooms were determined for evaluation of their dietary value. Na, K and Li contents were high, while Cr, Cu, Pb, Co, Zn, Mn and Ni concentration s were low in both Agaricus bisporus and Agaricus bitorquis. Proximate analysis showed that both mushrooms have sufficient quantity of protein, lipids, carbohydrates and cellulose. Gas chromatographic analysis of fatty acid methyl esters revealed that linoleic acid (44.19 %) and oleic acid (40.13 %) were the dominant fatty acids in Agaricus bisporus and Agaricus bitorquis respectively.

Assessment of Hydroponically Grown Macrophytes for Their Suitability as Fish Feed

Water hyacinth, water lettuce and parrot's feather plants were examined for their ability to remove nutrients from aquaculture wastewater at two retention times. During the experiment, the aquatic plants grew rapidly in the hydroponics system and appeared healthy with green color. At hydraulic retention times (HRTs) of 6 and 12 days, the average water hyacinth, water lettuce and parrot's feather yields were 83, 51 and 51 g (dm) m-2 and 49, 29 and 22 g (dm) m-2, respectively. The aquatic plants were able to significantly reduce the pollution load of the aquaculture wastewater. The TS, COD, NH4+-N, NO2--N, NO3--N and PO43--P reductions ranged from 21.4 to 48.0%, from 71.1 to 89.5%, from 55.9 to 76.0%, from 49.6 to 90.6%, from 34.5 to 54.4% and from 64.5 to 76.8%, respectively. Generally, the reductions increased with longer retention times and were highest in compartments containing water hyacinth followed by compartments containing water lettuce and parrot's feather. The nutritive value of the three wastewater grown plants was assessed to determine the suitability of using the plants as a component in fish feed. The three wastewater grown plants did not contain sufficient amounts of protein and fat to meet the dietary requirements of fish and shellfish. They also contained high concentrations of K, Cu, Fe, Mn, Se and Zn, which can lead to reduced feed intake, weight gain and growth rates in fish and shellfish.

Framework for the Rapid Optimization of Soluble Protein Expression in Escherichia coli Combining Microscale Experiments and Statistical Experimental Design

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

The Nutritive Value of Wastewater Grown Barley and its Utilization in Fish Feed

The feasibility of using wastewater grown barley plants as a component of fish feed was evaluated. The barley plants were grown in a hydroponics system on wastewater from a recirculating aquaculture facility. The effects of wastewater application rate on plant growth and pollution potential reduction were investigated. At the end of the experiment, the average crop heights and yields were 31.0 and 36.0 cm and 59 and 83 t ha-1 at wastewater application rates of 690 and 1380 mL compartment-1 day-1, respectively. The hydroponics system reduced the TS, COD, NH4+-N, NO2--N, NO3--N and PO4 -3-P of the aquaculture wastewater by 51.5-52.9, 72.3-72.3, 81.8-82.3, 97.9-98.2, 78.9-79.7 and 84.7-86.3%, respectively. The aquaculture wastewater grown barley met the energy, fat, Ca, Mg, P, Na, S and Mn dietary requirements of aquatic animals. It exceeded the carbohydrate, crude fiber, Cl, K, Cu, Fe, Se and Zn dietary requirements of fish and shellfish. It did not contain sufficient amounts of protein. The aquaculture wastewater grown barley could potential be used as a component in fish feed, but will require supplementation with a high protein source that contains low concentrations of carbohydrate, crude fiber, Cl, K, Cu, Fe, Se and Zn. Common protein sources that could be used for supplementation included fishmeal, bone meal and blood meal.

Automation and Optimization of Protein Expression and Purification on a Novel Robotic Platform

This article describes a novel robotic system for protein expression and purification. This area has traditionally proved a bottleneck in pharmaceutical discovery owing to the difficulty and unpredictability of outcome in evaluating multiple sets of expression conditions that will yield biologically active protein in sufficient quantity to meet the demands of discovery teams. This position has been exacerbated by the requirement of structural biology groups to have access to 100 mg scale quantities of pure protein for early and complex structural studies. The Piccolo robotic system described here was able to perform multiple parallel induction, expression, and single stage protein purification of a target protein (6-His tagged β-galactosidase) using a model E. coli expression system. The Piccolo system monitored E. coli cell growth automatically and rescheduled tasks dynamically based on individual instances of cell growth within purpose built 24-well bioreactor units termed culture vessel blocks. On completion of protein expression, the system performed cell lysis and purification of the expressed β-galactosidase through Ni-NTA histidine affinity chromatography. We show that alterations in the induction and expression conditions used resulted in changes in the yield of expressed β-galactosidase. In addition to the E. coli studies described here, the system is also applicable for other bacterial species and for eukaryotic cell culture applications. The Piccolo robotic system has the capacity to allow exploration of hundreds of different combinations of expression variables within a working week thereby allowing rapid identification of optimal conditions for expressing the biologically active protein of interest. This strategy confers a significant increase in the parallel processing capability of protein expression groups and goes a long way to addressing the current protein expression bottleneck.

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of ∼1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.

Effect of water-soluble fraction of cigarette smoke on human aortic endothelial cells – a proteomic approach

Proteomic analysis is an important investigative tool used to systematically explore cellular proteins that are responsive to adverse environmental challenges. Tobacco smoking is the second major cause of death in the world. In this study, we utilized two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) technologies to explore protein changes in human aortic endothelial cells (HAECs) in response to cigarette smoke extracts (CSE). Among 389 individual proteins resolved using 2-DE, 43 had a 2- to 3-fold change in levels as measured by spot intensity and 32 had more than a 3-fold change. Sixteen of the 32 spots with sufficient amount of proteins were excised for identification by performing matrix-assisted laser desorption/ionization (MALDI)-MS analysis. Using a peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified all these 16 proteins, which were either increased (n = 9) or decreased (n = 7) after CSE treatment. All these proteins have known functions, however, none have been reported to be altered after CSE treatment. The findings from our study suggest that utilizing a systemic investigative tool, such as the proteomic approach using 2-DE, may play an important role in discovering novel molecular mechanisms for cigarette smoking-induced pathological changes. Further investigation following the systemic discoveries must be further examined as they may potentially lead to new therapeutic approaches to smoking-induced diseases - a health issue affecting everyone in the world.

T cell responses to natural human proteins in primary biliary cirrhosis.

The study of T cell responses to autoantigens in human autoimmunity has been hampered by difficulties, firstly in identifying significant autoantigens, and secondly in the purification of authentic human proteins in sufficient quantities to allow characterization of antigen-specific T cell responses. In this study we have purified a human autoantigen, pyruvate dehydrogenase, retaining its enzymatic activity, and characterized autoreactive T cell responses to it in a human autoimmune disease, primary biliary cirrhosis. T cell responses to a mixture of the E2 and protein X subunits of human pyruvate dehydrogenase complex are seen in most affected patients, but in only a small minority of normal and chronic liver disease controls. By contrast, responses to whole pyruvate dehydrogenase complex occur with equal frequency in both groups. This suggests that responses to the E2 component/protein X of pyruvate dehydrogenase complex play a role in the pathogenesis of primary biliary cirrhosis. The availability of significant quantities of the human autoantigen in primary biliary cirrhosis makes this condition an interesting model in which to study true autoreactive human T cell responses.

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than 65 μM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

Application of In Vitro Protein Expression to Human Prote

INTRODUCTION. A critical step in many proteomics projects is the identification of genes that express proteins in sufficient quantities for subsequent studies. LLNL houses a collection of >200,000 human cDNAs, known as the IMAGE Consortium (http://image.llnl.gov)[1], including some full-length enriched cDNA libraries. We have developed an integrated system that exploits coupled in vitro transcription/translation (IVT) to identify human IMAGE cDNAs that express proteins of the predicted mass. Moreover, this expression screening can be conducted without plasmid DNA preparation or subcloning. IVT-produced proteins are used directly in functional assays or the cDNAs are transported to a bacterial expression system for large-scale protein production.

Cancer therapy by gene therapy with angiostatin.

Angiostatin, an internal fragment of plasminogen, has been shown to inhibit angiogenesis. A new area of cancer research that has generated excitement is the use of angiostatin to treat cancer. Angiostatin protein therapy has not been pursued because current technology is inadequate to manufacture the needed biologically active proteins in sufficient quantities. It is sufficient for effective therapy with angiostatin to establish angiostatin production in the vicinity of tumors by gene transfer of angiostatin cDNA. There are various methods by which to transfer angiostatin cDNA. One way is to use a viral vector to incorporate the gene into cells. Another way is to use nonviral vectors. In this review, evidence accumulated from many laboratories suggests that angiostatin gene therapy may be an important new cancer therapy as an adjuvant therapy to prevent recurrence. (c) 2001 Prous Science. All rights reserved.

Transient-expression technologies, their application and scale-up: 5-HT3 serotonin receptor case study

We have expressed the 5-HT3 receptor with two transient expression sytems: the Semliki Forest virus and the HEK293EBNA. Both systems allow the rapid expression of functional receptor protein at very high level: up to 107 receptors per cell. Large scale production yielded 1 to 2 mg of receptor protein per liter. The solubilisation of the receptor from membranes has been optimised; C12E9 solubilised 50 to 60 % of the ligand binding activity. A C-terminally engineered hexahistidine-tag facilited efficient purification of the receptor in a functional form. Sufficient quantities of protein are now available to enable biophysical characterisation of the 5-HT3 receptor.

Overproduction of SM28GST in a Baculovirus Expression Vector and Its Use to Evaluate the In vivo Immune Responses of Mice Vaccinated against Schistosoma mansoni with Naked DNA Encoding the SM28GST Gene

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing.

Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.

Gene expression systems in the development of high-throughput screens

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Purification and Characterization of the Bcl-2 Protein

The oncogene product bcl-2 functions as a repressor of programmed cell death and is a 26-kDa protein with a single predicted transmembrane segment located at the carboxyl terminus. The bcl-2 protein seems to function in different subcellular compartments, as evidenced by several biochemical and ultrastructural studies. The present study was performed to purify bcl-2 protein in significant quantities necessary for structural and functional studies. For this purpose, the bcl-2 gene was overexpressed in either baculovirus system or lymphocytes. Initially, attempts were undertaken to purify bcl-2 protein using conventional methods such as ion exchange or gel filtration chromatography. During these purification attempts we determined that bcl-2 protein is highly hydrophobic and prone to aggregation as might be expected for an integral membrane protein. By ion exchange and gel filtration chromatography, this protein could be partially purified. In order to purify bcl-2 to apparent homogeneity and avoid the aggregation problem, we prepared immunoaffinity columns using a monoclonal antibody developed against a synthetic peptide chosen from residues 61-76 of the amino acid sequence of human bcl-2. The antibody was either coupled to CNBr-activated Sepharose 4B or cross-linked into protein A-Sepharose by dimethylpimelimidate dihydrochloride. Cellular extract equivalent to 10 8 bcl-2-overexpressing insect cells or lymphocytes was applied to immunoaffinity columns. Approximately 500 μg purified bcl-2 protein could be recovered as estimated by silver staining and immunoblotting. Furthermore, purified bcl-2 protein was electroporated into Pre-B lymphocytes which do not express this protein in sufficient quantity to delay the onset of glucocorticoid-induced apoptosis. Following electroporation of homogeneously pure bcl-2 protein, the cells were found to prolong cell survival in response to glucocorticoids.