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We previously characterized VE-statin/egfl7, a protein that is exclusively secreted by endothelial cells and modulates smooth muscle cell migration. Here, we show that VE-statin/egfl7 is the first known natural negative regulator of vascular elastogenesis. Transgenic mice, expressing VE-statin/egfl7 under the control of keratin-14 promoter, showed an accumulation of VE-statin/egfl7 in arterial walls where its presence correlated with an impaired organization of elastic fibres. In vitro, fibroblasts cultured in the presence of VE-statin/egfl7 were unable to deposit elastic fibres due to a deficient conversion of soluble tropoelastin into insoluble mature elastin. VE-statin/egfl7 interacts with the catalytic domain of lysyl oxidase (LOX) enzymes and, in endothelial cells, endogenous VE-statin/egfl7 colocalizes with LoxL2 and inhibits elastic fibre deposition. In contrast, mature elastic fibres are abundantly deposited by endothelial cells that are prevented from producing endogenous VE-statin/egfl7. We propose a model where VE-statin/egfl7 produced by endothelial cells binds to the catalytic domains of enzymes of the LOX family in the vascular wall, thereby preventing the crosslink of tropoelastin molecules into mature elastin polymers and regulating vascular elastogenesis.  相似文献   

5.
Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).  相似文献   

6.
Epidermal growth factor (EGF) is a protein that belongs to the family of growth factors that bind the ErbB receptors, which play a prominent role in the development of carcinomas. We had demonstrated that potato carboxypeptidase inhibitor (PCI) acts as an EGF antagonist. Because of the low affinity of PCI for the epidermal growth factor receptor, it was decided to design EGF mutants with PCI abilities. In order to achieve this we have first cloned, expressed and purified the native protein, EGF. Different expression systems with different locations of the recombinant protein were designed and a purification protocol was designed with those which allowed expression of EGF. Finally, the sample needed folding. Differences in the amount of EGF obtained and its activity were observed depending on the expression system used.  相似文献   

7.
Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues.  相似文献   

8.
The immunoglobulin superfamily protein neurolin plays a central role during differentiation and development of retina ganglion cells in goldfish. As shown in earlier work, blockage of the second immunoglobulin domain (Ig2) of neurolin with domain-specific antibodies causes severe pathfinding defects of growing axons in the retina. Thus Ig2 of neurolin was identified as the critical domain for axon guidance. In the present study we have developed a protocol for expression and purification of neurolin-Ig2 suitable for structure analysis, functional studies and ligand identification. Neurolin was expressed in Rosettagami and Origami strains of Escherichia coli which is deficient in glutathione- and thioredoxin reductase facilitating proper formation of the disulfide bond in the cytoplasm. The protein was purified via an N-terminal His(6)-tag by Ni(2+) affinity and size exclusion chromatography. After purification the His(6)-tag was cut-off without loss of solubility. Analytical size exclusion chromatography revealed an apparent molecular mass for neurolin-Ig2 in agreement with a non-covalent homodimer. Analysis of CD and FTIR spectra gave a secondary structure content typical for Ig domains.  相似文献   

9.
The purification of pp60c-src has been hampered by the low levels of protein it represents in most cells and its tendency to undergo proteolysis during purification. The discovery that the platelet expresses unusually high levels of pp60c-src has made large-scale purification from a normal source feasible. We have developed a method for the purification of intact pp60c-src to near homogeneity from human platelets and have determined the enzymatic properties of this purified protein in vitro. Rapid, high yield purification of pp60c-src from isolated platelet membranes was achieved in a two-step protocol involving sequential chromatography on an anti-pp60c-src immunoaffinity matrix and phenyl-Sepharose. This protocol yielded 0.5 mg of pp60c-src from 30 units of platelets. Using enolase as an exogenous substrate, the specific activity of the enzyme was 25 nmol P.min-1.mg-1. The Km for MnATP2- for enolase phosphorylation (2.2 microM) was higher than for the autophosphorylation of pp60c-src (0.6 microM). Maximal enzyme activity required either Mn2+ or Mg2+, and both ATP and GTP could be utilized as the phosphate donor. Evidence is shown which indicate that the autophophorylation of pp60c-src in vitro occurs through an intramolecular mechanism and that this reaction is reversible.  相似文献   

10.
Due to their involvement in a variety of physiological and pathological processes, different isoforms of annexins are being utilized as markers of some human diseases and bio-imaging of tissue injury (due to apoptosis), and have been proposed as drug delivery vehicles. These, in addition to extensive biophysical studies on the role of annexins in organizing lipid domains in biological membranes, have necessitated development of an efficient protocol for producing annexins in bulk quantities. In this paper, we report a one-step purification protocol for annexin a5 without using lipid vesicles or involving any column chromatographic step. Depending on the growth and expression condition, a fraction of recombinant annexin a5 (cloned in pET3d vector) was sequestered into inclusion bodies. When these inclusion bodies were dissolved in 6 M urea, subjected to a 10-fold snap dilution in the presence of 5 mM Ca(2+) and stored overnight at 4 degrees C, annexin a5 was precipitated as a homogenous protein as judged by SDS-PAGE. This one-step purification protocol produced about 35 mg of highly purified annexin a5 per liter of bacterial culture. The annexin a5 purified from inclusion bodies exhibited similar properties to that obtained from the soluble fraction using the conventional lipid-partitioning approach. Our purification protocol for annexin a5 elaborated herein is equally effective for purification of annexin A2, and we believe, will serve as general protocol for purifying other annexins in bulk quantities for diagnostic as well as detailed biophysical studies.  相似文献   

11.
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.  相似文献   

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Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.  相似文献   

14.
We developed a protocol for the fast purification of small proteins and peptides using heat incubation as the first purification step. The proteins are expressed from a new bacterial expression vector (pETM-90) fused to the C-terminus of thermostable Ftr from Methanopyrus kandleri. The vector further contains a 6xHis-tag to allow immobilised metal ion affinity purification and a TEV protease cleavage site to enable the removal of the His-tag and fusion partner. Heat incubation induces the specific denaturation and precipitation of the Escherichia coli proteins but not of the thermostable fusion protein. Using the fusion construct and the heat incubation protocol a number of fusion proteins were purified to near homogeneity. The thermostability was ensured when Ftr had a molecular weight higher than twice the target protein. The obtained purification yields were similar and, in some cases, even higher than the ones obtained by affinity purification with the same Ftr-fusion proteins or the same target proteins fused to other often used partners such as NusA, GST, or DsbA. The protocol does not depend on a specific thermostable protein as was shown by the exchange of Ftr for M. kandleri Mtd. Purification by heat incubation is a fast and inexpensive alternative to chromatographic techniques, particularly suitable for the production of antigenic sequences for which the loss of native structure is not detrimental. We proved that it can be easily automated.  相似文献   

15.
Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.  相似文献   

16.
We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.  相似文献   

17.
The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.  相似文献   

18.
We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.  相似文献   

19.
Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.  相似文献   

20.
As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.  相似文献   

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