Heparan sulfates targeting increases MHC class I- and MHC class II-restricted antigen presentation and CD8(+) T-cell response.

Heparan sulfates (HS) are carbohydrate moieties of HS proteoglycans (HSPGs). They often represent alternative attachment points for proteins or microorganisms targeting receptors. HSPGs, which are ubiquitously expressed, thereby participate in numerous biological processes.

Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.

Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork.

A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21.

Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers.

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.

BgK, a disulfide-containing sea anemone toxin blocking K+ channels, can be produced in Escherichia coli cytoplasm as a functional tagged protein.

BgK, a sea anemone peptide consisting of 37 amino acid residues and 3 disulfide bonds, blocks voltage-gated potassium (Kv1) channels. Here, we report a method for producing tagged BgK in Escherichia coli, as a soluble cytoplasmic protein. First, using peptidic synthesis, we show that addition of a 15 residue peptide (S.

Expression of recombinant alkaline phosphatase conjugates in Escherichia coli.

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA.

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment.

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding.

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state.

Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket.

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation.

Do structural deviations between toxins adopting the same fold reflect functional differences?

Three-finger proteins form a structurally related family of compounds that exhibit a great variety of biological properties. To address the question of the prediction of functional areas on their surfaces, we tentatively conferred the acetylcholinesterase inhibitory activity of fasciculins on a short-chain curaremimetic toxin. For this purpose, we assimilated the three-dimensional structure of fasciculin 2 with the one of toxin alpha.

Molecular and structural basis of the specificity of a neutralizing acetylcholine receptor-mimicking antibody, using combined mutational and molecular modeling analyses.

The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin-specific monoclonal antibody called Malpha2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the Malpha2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp(31) in complementarity determining region 1H is close to, and perhaps interacts with, Arg(33) in the antigen.

Stability of a structural scaffold upon activity transfer: X-ray structure of a three fingers chimeric protein.

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones.

Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N.

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv).

Increasing immunogenicity of antigens fused to Ig-binding proteins by cell surface targeting.

Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety.

Transformation of a non-enzymatic toxin into a toxoid by genetic engineering.

Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies.

Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?

Recent studies revealed that animal toxins with unrelated biological functions often possess a similar architecture. To tentatively understand the evolutionary mechanisms that may govern this principle of functional prodigality associated with a structural economy, two complementary approaches were considered. One of them consisted of investigating the rates of mutations that occur in cDNAs and/or genes that encode a variety of toxins with the same fold.

The functional architecture of an acetylcholine receptor-mimicking antibody.

Malpha2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its three-dimensional structure has been previously predicted by molecular modeling, suggesting that 29 complementarity determining region (CDR) residues and 2 framework residues are exposed to solvent. To identify the antibody residues that bind to the antigen, i.

High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B.

Complete amino-acid sequence of the beta-subunit of VTX from venom of the stonefish (Synanceia verrucosa) as identified from cDNA cloning experiments.

A cDNA encoding a subunit of the verrucotoxin (VTX) has been identified from a cDNA library derived from stonefish venom glands. It encodes a polypeptide of 708 amino-acid residues, followed by a 3'-untranslated region of 895 bp long. The ORF contains the complete mature sequence of the beta-subunit of the VTX, as inferred from both the presence of an identical N-terminus sequence and 96% homology among the 506 amino terminus residues found in the partial sequence of the beta-subunit of the stonustoxin from Synanceia horrida (Ghadessy, F.

Mimicry between receptors and antibodies. Identification of snake toxin determinants recognized by the acetylcholine receptor and an acetylcholine receptor-mimicking monoclonal antibody.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3.

Engineering of a recombinant colorimetric fusion protein for immunodiagnosis of insulin.

A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene. A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene. The recombinant conjugate was stable and could be produced in the bacteria.

A recombinant insect-specific alpha-toxin of Buthus occitanus tunetanus scorpion confers protection against homologous mammal toxins.

We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an alpha-toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig-binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant apha-toxin/l bacterial culture.