共查询到20条相似文献,搜索用时 31 毫秒
1.
Neumeyer J Hallas C Merkel O Winoto-Morbach S Jakob M Thon L Adam D Schneider-Brachert W Schütze S 《Experimental cell research》2006,312(11):2142-2153
The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI DeltaTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI DeltaTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI DeltaTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis. 相似文献
2.
K Dahlman P E Str?mstedt C Rae H J?rnvall J I Flock J Carlstedt-Duke J A Gustafsson 《The Journal of biological chemistry》1989,264(2):804-809
A fragment comprising the DNA-binding domain of the human glucocorticoid receptor has been expressed in a functional form in Escherichia coli as a fusion protein with protein A from Staphylococcus aureus. The DNA-binding domain was purified to apparent homogeneity by affinity chromatography on IgG-Sepharose and DNA-cellulose, a purification scheme which does not involve denaturation of the protein at any step. The DNA-binding domain was separated from the protein A part of the fusion protein by domain-specific enzymatic cleavage with chymotrypsin while immobilized on IgG-Sepharose. The recombinant protein has been characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reactivity to iodoacetate and was found to correspond to the primary structure derived from the cDNA sequence. DNase I footprinting showed that the purified recombinant protein bound to the same DNA sequences on the mouse mammary tumor virus long terminal repeat as glucocorticoid receptor purified from rat liver does. About 10 times more recombinant protein, on a molar basis, was needed to obtain the same level of protection. However, the protection of the three different footprints (1.3, 1.4, and 1.5') by the recombinant protein differed greatly from that of the natural receptor, with virtually no protection of footprint 1.4. This indicates cooperative binding of the natural receptor to adjacent footprints, dependent on other regions of the receptor than the DNA-binding domain. 相似文献
3.
Purification of the receptor for platelet-derived growth factor from porcine uterus 总被引:15,自引:0,他引:15
L R?nnstrand M P Beckmann B Faulders A Ostman B Ek C H Heldin 《The Journal of biological chemistry》1987,262(7):2929-2932
The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor. 相似文献
4.
G De Wilde J Murray-Rust E Boone D Olerenshaw N Q McDonald C Ibanez G Haegeman A Wollmer M Federwisch 《European journal of biochemistry》2001,268(5):1382-1391
Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference. 相似文献
5.
The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest classes of protein interaction modules and plays a pivotal role in the apoptosis, inflammation, and immune cell signaling pathways. Despite the biological importance of the death domain superfamily, structural and in vitro biochemical studies have been limited because these domains are prone to aggregate under physiological conditions. Here, we describe a generalized method, termed semi-refolding, that is particularly applicable for purification of the functional death domain superfamily. The recombinant proteins Caspase-1 CARD, AIM2 PYD, NALP3 PYD, and RIP1 DD from inclusion bodies were successfully purified using this method. 相似文献
6.
To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies. 相似文献
7.
In the present study, calf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5 while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa. 相似文献
8.
D. van Loon M.M. Voorhorst A.O. Brinkmann E. Mulder 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1988,970(3)
In the present study, culf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5, while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa. 相似文献
9.
One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit 总被引:13,自引:0,他引:13
A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG
Claes Ingmar JJ Segers Marijke E Verhoeven Tine LA Dusselier Michiel Sels Bert F De Keersmaecker Sigrid CJ Vanderleyden Jos Lebeer Sarah 《Microbial cell factories》2012,11(1):1-8
Background
Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).Result
The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.Conclusions
These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies. 相似文献11.
The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCAID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn2+ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn2+ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases. 相似文献
12.
5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is built of a catalytic C-terminal domain and a regulatory N-terminal C2-like domain. The C2-like domain is the target of many regulatory factors or proteins including Ca(2+), phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-LO by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. In order to obtain the 5-LO C2-like domain as purified protein in good yields for further mechanistic studies and structure elucidation, a novel expression and purification approach has been applied. A plasmid was constructed expressing a fusion protein of maltose-binding protein (MBP) and the regulatory C2-like domain of 5-LO (AS 1-128), separated by a tobacco etch virus (TEV) protease-cleavage site. The fusion protein MBP-5LO1-128 could be essentially expressed as a soluble protein in Escherichia coli and was efficiently purified by amylose affinity chromatography. By means of this procedure, approximately 80mg purified fusion protein out of 1L E. coli culture were obtained. Digestion with TEV protease yielded the C2-like domain that was further purified using hydrophobic interaction chromatography. Alternatively, the uncleaved fusion protein MBP-5LO1-128 may be suitable to immobilize the C2-like domain on an amylose resin for co-factor interaction studies. Together, we present a convenient expression and purification strategy of the 5-LO C2-like domain that opens many possibilities for structural determination and mechanistic studies, aiming to reveal the precise role and function of this regulatory domain. 相似文献
13.
Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli 总被引:1,自引:0,他引:1
Parkar AA Stow MD Smith K Panicker AK Guilloteau JP Jupp R Crowe SJ 《Protein expression and purification》2000,20(2):152-161
We have cloned, overexpressed, and purified the catalytic domain (residues Gly106 to Asn268) of human macrophage metalloelastase (MMP-12) in Escherichia coli. This construct represents a truncated form of the enzyme, lacking the N-terminal propeptide domain and the C-terminal hemopexin-like domain. The overexpressed protein was localized exclusively to insoluble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclusion bodies were solubilized in an 8 M guanidine-HCl buffer and purified by gel filtration chromatography under denaturing conditions. Partial refolding of the protein by dialysis into a 3 M urea buffer caused selective degradation of the truncated form of the protein, while the intact catalytic domain was unaffected by proteolysis. An SP-Sepharose chromatography step purified the protein to homogeneity and served also to complete the refolding. The purified protein was homogeneous by mass spectrometry and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a concentration of 9 mg/ml. This purification procedure enables the production of 23 mg of protein per liter of E. coli culture and is amenable to large-scale protein production for structural studies. 相似文献
14.
Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system 总被引:6,自引:0,他引:6
R Herrera D Lebwohl A Garcia de Herreros R G Kallen O M Rosen 《The Journal of biological chemistry》1988,263(12):5560-5568
The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system. A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection. The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro. Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein. Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor). Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151). Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain. This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation. The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity. These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase. 相似文献
15.
Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core. 相似文献
16.
Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit 总被引:3,自引:0,他引:3
The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit. 相似文献
17.
Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor 总被引:3,自引:0,他引:3
Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated. 相似文献
18.
Li Q Gayen S Chen AS Huang Q Raida M Kang C 《Biochemical and biophysical research communications》2010,403(1):126-132
The human Ether-à-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker. 相似文献
19.
Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein,Endo180 总被引:1,自引:0,他引:1
Rivera-Calzada A Robertson D MacFadyen JR Boskovic J Isacke CM Llorca O 《EMBO reports》2003,4(8):807-812
Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by single-particle electron microscopy to obtain a three-dimensional structure of the N-terminal part of the protein at a resolution of 17 Å and reveal, for the first time, the interactions between non-adjacent domains in the mannose receptor family. We show that for Endo180, the cysteine-rich domain contacts the second C-type lectin-like domain, thus providing structural insight into how modulation of its several ligand interactions may regulate Endo180 receptor function. 相似文献
20.
The plant hormone cytokinin is implicated in a large number of developmental and physiological processes. In the model plant Arabidopsis thaliana cytokinin is perceived by a class of membrane-bound receptor histidine kinases with three members, namely AHK2, AHK3, and CRE1/AHK4. These receptors possess an N-terminally located putative extracellular cyclases/histidine kinases associated sensor extracellular (CHASE) domain, which is responsible for hormone recognition. This hydrophilic domain and the two flanking transmembrane regions (CHASE-TM) were expressed using a cell-free protein expression system based on a bacterial ribosomal extract. To obtain soluble CHASE-TM protein, different detergents were directly added to the cell-free reaction and their effect on the yield of soluble protein was studied. After optimising the experimental set-up and employing Brij 58 as a detergent more than 3 mg/ml soluble protein of the CHASE-TM domain were obtained. Affinity purification via a C-terminally fused His-tag resulted in greater than 90% purity. The identity of the purified domain was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Circular dichroism spectroscopy was used and a predominantly α-helical folding pattern was shown, which is in good accordance with secondary structure prediction. A newly developed cytokinin binding assay confirmed the functionality of the thus expressed and purified CHASE-TM domain. The work presented clearly demonstrates the feasibility of producing high amounts of a plant membrane protein using a cell-free protein expression system. This opens the possibility of further biochemical and pharmacological analysis, as well as structural studies on this type of receptor protein. 相似文献