scholarly journals Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

1989 ◽  
Vol 109 (2) ◽  
pp. 593-605 ◽  
Author(s):  
M Way ◽  
J Gooch ◽  
B Pope ◽  
A G Weeds
Keyword(s):  
Escherichia Coli ◽  
Human Plasma ◽  
Binding Sites ◽  
Current Knowledge ◽  
Limited Proteolysis ◽  
Actin Binding ◽  
High Yield ◽  
Soluble Form ◽  
Proteolytic Fragment ◽  
Plasma Gelsolin

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.

scholarly journals The actin filament-severing domain of plasma gelsolin.

1986 ◽  
Vol 103 (4) ◽  
pp. 1473-1481 ◽  
Author(s):  
C Chaponnier ◽  
P A Janmey ◽  
H L Yin
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
The Other ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Chymotryptic Peptide ◽  
Native Plasma

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

scholarly journals Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.

1988 ◽  
Vol 106 (3) ◽  
pp. 805-812 ◽  
Author(s):  
H L Yin ◽  
K Iida ◽  
P A Janmey
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Assembly ◽  
Plasma Gelsolin ◽  
High Affinity ◽  
Binding Data ◽  
Actin Binding Domain

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.

scholarly journals Identification of fibronectin fragments that bind to carboxy-group-modified proteins

1983 ◽  
Vol 215 (3) ◽  
pp. 613-616 ◽  
Author(s):  
M Vuento ◽  
K Sekiguchi ◽  
M Korkolainen
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Carboxy Group ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Plasma Fibronectin ◽  
C Terminus ◽  
Fibronectin Fragments ◽  
Modified Proteins ◽  
Polypeptide Chains

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.

Purification and crystallization of a proteolytic fragment of Escherichia coli pyruvate formate-lyase

1999 ◽  
Vol 55 (2) ◽  
pp. 531-533 ◽  
Author(s):  
Veli-Matti Leppänen ◽  
Camran V. Parast ◽  
Kenny K. Wong ◽  
John W. Kozarich ◽  
Adrian Goldman
Keyword(s):  
Escherichia Coli ◽  
Limited Proteolysis ◽  
X Ray Diffraction ◽  
Anaerobic Conditions ◽  
Protein Fragment ◽  
Proteolytic Fragment ◽  
Pyruvate Formate Lyase ◽  
X Ray ◽  
Cell Dimensions ◽  
Polyethylene Glycol 1000

Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyase (PFL) is the first reaction after the production of pyruvate in the glycolytic pathway. Crystallization trials with Escherichia coli PFL were unsuccessful and therefore limited proteolysis was used to produce a stable crystallizable N-terminal protein fragment by trypsin cleavage. The molecular weight of this cleavage product was found to be 69.6 kDa by MALDI MS analysis, and the DNA sequence corresponding to this fragment was cloned. The recombinant protein fragment was crystallized by sitting-drop vapour diffusion using polyethylene glycol 1000 as precipitant. The crystals, which grew to 2 mm in length and 0.2 mm in cross section, belong to the hexagonal space group P61 or P65 with cell dimensions a = b = 140.4, c = 215.3 Å and two molecules per asymmetric unit. X-ray diffraction data were collected from 20 to 3.2 Å resolution from a single frozen crystal on a synchrotron-radiation beamline.

scholarly journals Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus

2004 ◽  
Vol 382 (2) ◽  
pp. 667-675 ◽  
Author(s):  
Hiroaki TATENO ◽  
Harry C. WINTER ◽  
Irwin J. GOLDSTEIN
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Expression System ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Soluble Form ◽  
Carbohydrate Binding ◽  
Terminal Portion ◽  
Sugar Binding ◽  
Polyporus Squamosus

Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acα2,6Galβ1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)3 domain at its N-terminus that is composed of three homologous subdomains (α, β and γ). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acα2,6Galβ1,4GlcNAc, but not for the sialyl α2,3-linked isomer. We also investigated the structure–function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectin's molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the β and γ subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acα2,6Galβ1,4GlcNAc sequence of asparagine-linked glycans.

scholarly journals Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface

2005 ◽  
Vol 385 (3) ◽  
pp. 659-665 ◽  
Author(s):  
Ulrike FOCK ◽  
Brigitte M. JOCKUSCH ◽  
Wolf-Dieter SCHUBERT ◽  
Horst HINSSEN
Keyword(s):  
Tertiary Structure ◽  
Limited Proteolysis ◽  
Cell Types ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Equine Plasma ◽  
Binding Epitope ◽  
N Terminus ◽  
Immunological Approach ◽  
Cytoplasmic Gelsolin

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34–Ile45 and Leu64–Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli

2020 ◽  
Vol 27 (3) ◽  
pp. 210-218
Author(s):  
Bahador Bakhtiarvand ◽  
Zahra Sadeghi ◽  
Shirin Tarahomjoo ◽  
Soheila Yaghmaie
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Salmonella Enterica ◽  
Inclusion Bodies ◽  
Vaccine Development ◽  
Limited Proteolysis ◽  
Soluble Form ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Native Page ◽  
Chemical Chaperones

Background: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. Objective: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. Methods: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. Results: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. Conclusion: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.

scholarly journals Soluble Expression of Small Antibody Fragments against PD-L1 Using Escherichia coli with High Yield and Purity

2021 ◽  
Vol 11 (19) ◽  
pp. 9149
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Escherichia Coli ◽  
Tumor Cells ◽  
Surface Protein ◽  
Antibody Fragments ◽  
High Yield ◽  
Soluble Form ◽  
Single Chain ◽  
High Penetration ◽  
Variable Heavy ◽  
Variable Light

Programmed death-ligand 1 (PD-L1) is a surface protein overexpressed in tumor cells. Recently, targeted therapy using PD-L1 antibodies to reconstitute the antitumor activity of T cells has received considerable attention as a cancer treatment. Among the several types of anti-PD-L1 antibodies, small-sized antibody fragments are useful agents to block PD-L1 for experimental and therapeutic purposes owing to their high penetration efficacy toward dense tumor cells. Herein, we expressed and purified recombinant single chain fragment of variable domain, variable heavy chain, and variable light chain, against PD-L1 in a soluble form using Escherichia coli, resulting in their high yield and high purity. We confirmed the antigen-binding efficiency of these antibody fragments, which showed antigen concentration-dependent responses. These results suggest that these small antibody fragments can serve as new agents for blocking or detecting PD-L1.

scholarly journals Microinjection of gelsolin into living cells.

1987 ◽  
Vol 104 (3) ◽  
pp. 491-501 ◽  
Author(s):  
J A Cooper ◽  
J Bryan ◽  
B Schwab ◽  
C Frieden ◽  
D J Loftus ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Calcium Concentration ◽  
Simple Procedure ◽  
Actin Binding ◽  
Proteolytic Fragment ◽  
Plasma Gelsolin ◽  
Terminal Fragment ◽  
Nucleation Activity ◽  
The Difference

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

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