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.

The melibiose/Na + symporter of Escherichia coli : kinetic and molecular properties

1990 ◽  
Vol 326 (1236) ◽  
pp. 411-423 ◽  
Keyword(s):  
Escherichia Coli ◽  
Complex Cation ◽  
Expression System ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Coupling Mechanism ◽  
Facilitated Diffusion ◽  
Histidine Residues ◽  
Sugar Binding ◽  
Dependent Transport

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H + , Na + or Li + -sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar—carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

scholarly journals Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis

2018 ◽  
Author(s):  
Julie E. Heggelund ◽  
Joel B. Heim ◽  
Gregor Bajc ◽  
Vesna Hodnik ◽  
Gregor Anderluh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Enterotoxigenic Escherichia Coli ◽  
Resonance Spectroscopy ◽  
Carbohydrate Binding ◽  
Heat Labile ◽  
Causes Of Mortality ◽  
Site Mutagenesis ◽  
Toxin Uptake ◽  
Great Burden

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galb4GlcNAcb6[Galb4GlcNAcb3]Galb4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding, enhancing avidity.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

scholarly journals Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli: Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

2010 ◽  
Vol 76 (9) ◽  
pp. 2729-2739 ◽  
Author(s):  
Avijit Biswas ◽  
Yasmin M. Vasquez ◽  
Tierna M. Dragomani ◽  
Monica L. Kronfel ◽  
Shervonda R. Williams ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Expression System ◽  
Water Soluble ◽  
Soluble Form ◽  
Beta Subunits ◽  
Lyase Activity ◽  
Synechococcus Sp ◽  
Tetrapyrrole Chromophores

ABSTRACT Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and α-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter−1 of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (αAP-B) and ApcF (β18). The N-terminal, allophycocyanin-like domain of ApcE (LCM 99) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of β-phycocyanin.

scholarly journals Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

1995 ◽  
Vol 15 (10) ◽  
pp. 5627-5634 ◽  
Author(s):  
Z Weng ◽  
R J Rickles ◽  
S Feng ◽  
S Richard ◽  
A S Shaw ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Function Analysis ◽  
Binding Specificity ◽  
Sh3 Domain ◽  
Single Amino Acid ◽  
Sh3 Domains ◽  
Cell Extracts

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.

scholarly journals Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography

2019 ◽  
Author(s):  
Julie E. Heggelund ◽  
Joel B. Heim ◽  
Gregor Bajc ◽  
Vesna Hodnik ◽  
Gregor Anderluh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Enterotoxigenic Escherichia Coli ◽  
Resonance Spectroscopy ◽  
Carbohydrate Binding ◽  
Heat Labile ◽  
Causes Of Mortality ◽  
Site Mutagenesis ◽  
Toxin Uptake ◽  
Great Burden

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galbeta4GlcNAcbeta6[Galbeta4GlcNAcbeta3]Galbeta4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

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