Further characterisation of the talin-binding site in the cytoskeletal protein vinculin

1992 ◽  
Vol 103 (3) ◽  
pp. 719-731 ◽  
Author(s):  
A.P. Gilmore ◽  
P. Jackson ◽  
G.T. Waites ◽  
D.R. Critchley
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Binding Activity ◽  
Cytoskeletal Proteins ◽  
Cytoskeletal Protein ◽  
Glutathione S Transferase ◽  
Agarose Beads ◽  
Cell Matrix ◽  
Sds Page ◽  
Alternatively Spliced

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1–398, but not to those spanning residues 399–881 or 881–1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167–207. Interestingly, a fusion protein spanning residues 1–398, but lacking residues 167–207, was unable to bind talin. However, further analysis showed that residues 167–207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1–258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1–398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5′ portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167–207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.

Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli

2014 ◽  
Vol 998-999 ◽  
pp. 248-251
Author(s):  
Zhi Xin Di ◽  
Jian Zhong Ma ◽  
Yong Gang Wang
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Human Proinsulin ◽  
Sds Page ◽  
Dna Fragment ◽  
Sequence Encoding

A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

scholarly journals Cyclophilin-40: evidence for a dimeric complex with hsp90

1995 ◽  
Vol 307 (1) ◽  
pp. 5-8 ◽  
Author(s):  
K Hoffmann ◽  
R E Handschumacher
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Fusion Protein ◽  
Steroid Receptors ◽  
Heat Shock Protein 90 ◽  
Binding Activity ◽  
Dimeric Complex ◽  
Glutathione S Transferase ◽  
High Concentration ◽  
Cyclophilin 40

The expression of human cyclophilin 40 (CyP-40) as a glutathione S-transferase fusion protein has provided a means to identify cellular components that are in association with this ubiquitous protein. When the fusion protein was coupled to a GSH affinity matrix, heat-shock protein 90 (hsp90) was found to be the predominant associated protein in all tissue extracts examined. The relatively high concentration of each of these proteins in various tissues indicates that the dimeric complex exists in concentrations that exceed those of the inactive steroid receptors of which each protein is a component. Association does not occur with heat-shock protein 70 and is not affected by cyclosporin A (CsA). Independent expression of two domains of CyP-40 permitted dissociation of N-terminal isomerase and CsA binding activity from the hsp90 binding site, which is located at the FKBP-59-like C-terminal region. The biological association of CyP-40 with hsp90 in many tissues may reflect a conjoint role in protein folding and trafficking.

scholarly journals Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

1995 ◽  
Vol 182 (4) ◽  
pp. 1089-1099 ◽  
Author(s):  
K Tachibana ◽  
T Sato ◽  
N D'Avirro ◽  
C Morimoto
Keyword(s):  
Amino Acids ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fusion Proteins ◽  
Focal Adhesions ◽  
Immunohistochemical Analysis ◽  
Binding Activity ◽  
Glutathione S Transferase ◽  
Cytoskeleton Protein ◽  
Direct Association

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins.

1987 ◽  
Vol 105 (5) ◽  
pp. 2103-2110 ◽  
Author(s):  
T J Byers ◽  
R Dubreuil ◽  
D Branton ◽  
D P Kiehart ◽  
L S Goldstein
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Single Copy ◽  
Binding Activity ◽  
Bovine Brain ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Expression Library ◽  
Calmodulin Binding ◽  
Alpha Spectrin

Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.

scholarly journals Identification of the vinculin-binding site in the cytoskeletal protein α-actinin

1994 ◽  
Vol 301 (1) ◽  
pp. 225-233 ◽  
Author(s):  
A McGregor ◽  
A D Blanchard ◽  
A J Rowe ◽  
D R Critchley
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Fusion Proteins ◽  
Spherical Particles ◽  
Sedimentation Equilibrium ◽  
Actin Binding ◽  
Glutathione S Transferase ◽  
Agarose Beads ◽  
Shaped Particle ◽  
Particle Length

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both ‘lollipop’- and ‘dumbell’-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.

scholarly journals Aggregation of truncated GST--HD exon 1 fusion proteins containing normal range and expanded glutamine repeats

1999 ◽  
Vol 354 (1386) ◽  
pp. 991-994 ◽  
Author(s):  
B. Hollenbach ◽  
E. Scherzinger ◽  
K. Schweiger ◽  
R. Lurz ◽  
H. Lehrach ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Molecular Mass ◽  
Fusion Proteins ◽  
Amyloid Fibrils ◽  
Protein Aggregates ◽  
Proteolytic Cleavage ◽  
High Molecular Mass ◽  
Glutathione S Transferase ◽  
Exon 1 ◽  
Uniform Diameter

We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington'sdisease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al . (1997) Cell 90, 549-558 ). Here we report that a truncated GST-HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51ΔP) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca . 25 nm. In contrast, proteolytic cleavage of GST-HD51ΔP resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and Δ-amyloid fibrils in Alzheimer'sdisease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20ΔP and GST-HD30ΔP did not show any tendency to form more highly ordered structures, either with or without protease treatment.

scholarly journals Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-β-galactosidase fusion proteins

1990 ◽  
Vol 270 (2) ◽  
pp. 463-468 ◽  
Author(s):  
J C Brown ◽  
J H Spragg ◽  
G N Wheeler ◽  
P W Taylor
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Yolk Sac ◽  
Western Blots ◽  
C Terminus ◽  
Sds Page ◽  
A Subunit ◽  
B Subunits ◽  
Beta Galactosidase

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

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