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A Charbit

Publications and source records attributed to A Charbit.

At least 55 records · Page 3Linked to original sources
PubMed1990

Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli.

After fusion with the N-proximal portion of the outer membrane protein LamB, three beta-adrenergic receptors, the human beta 1- and beta 2- and turkey beta 1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three beta-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.

Animals↗
PubMed1990

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation.

Salmonella typhimurium and S. typhi were transformed with high efficiency by electroporation. Transformation efficiencies of up to 10(10) transformants per microgram of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a ga1E mutation slightly improved transformation efficiency in S. typhimurium (less than tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.

Antigens, Bacterial↗
PubMed1990

Critical role of neighbouring sequences on the immunogenicity of the C3 poliovirus neutralization epitope expressed at the surface of recombinant bacteria.

The C3 neutralization epitope of poliovirus type 1 (PV-1) is a continuous epitope comprised within residues 93-103 of capsid protein VP1. These residues form a loop at the surface of the virus particle. The authors compared the immunogenicity of two peptides which contain this epitope, when presented at the surface of Escherichia coli by genetic insertion in the outer membrane protein LamB. One peptide was 13 residues long (VP1:93-103) and the other one contained flanking sequences increasing its size to 35 residues (VP1:86-115). Mice and rabbits were immunized with recombinant bacteria expressing the corresponding LamB-VP1 hybrid proteins. Antibodies against synthetic peptides, against native and heat denatured viral particles, as well as neutralizing antibodies were monitored. In this mode of presentation the shortest form of the epitope was more immunogenic. We provide evidence that the conformation of the epitope is different in the two hybrid LamB proteins and discuss possible consequences for immunogenicity.

Amino Acid Sequence↗
PubMed1990

Immunogenicity and antigenicity of conserved peptides from the envelope of HIV-1 expressed at the surface of recombinant bacteria.

We expressed peptides from the HIV-1 envelope protein at the surface of Escherichia coli by genetic insertions into an exposed loop of the outer membrane protein LamB. Recombinant bacteria expressing eight peptides from gp110 (pep1-pep8), conserved between HIV-1 and HIV-2, were used as live immunogens in rabbits by the intravenous route. The eight constructions elicited anti-LamB antibodies, showing that the hybrid proteins were immunogenic. One of them, LamB-pep8, gave rise to antibodies able to react with gp160 and to neutralize HIV-1 in vitro. We also show that this type of recombinant E. coli can provide a convenient reagent to monitor and characterize specific antibodies. Recombinant clones were used to test sera of seropositive individuals, as well as to narrow down the monoclonal antibody 110-1 recognition site to a cluster of eight residues at the carboxy-terminal end of gp110.

Amino Acid Sequence↗
PubMed1989

Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12.

LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Antibody response to a foreign epitope expressed at the surface of recombinant bacteria: importance of the route of immunization.

A genetic procedure has been previously established to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier. A portion of the pre-S2 region of hepatitis B virus, residues 132-145, has been inserted at amino acid position 153 of the LamB protein, in a cell surface exposed loop. In the present study, we have analysed the antibody responses induced by these recombinant bacteria (live, heat-killed or sonicated) depending upon the route of immunization. The intravenous (i.v.) or intraperitoneal (i.p.) administration of the live recombinant bacteria to mice induced the synthesis of antibodies against both the inserted peptide and the native LamB protein. The antibodies raised recognized HBsAg particles. These mice also had high titres of antibodies against E. coli antigens (as determined using a crude bacterial sonicate). In contrast, mice immunized subcutaneously (s.c.) did not develop antibodies against the pre-S2 peptide nor against the HBsAg particles. Their anti-LamB responses were low compared with the response of mice immunized by the parenteral route. Interestingly, s.c. or i.v. immunizations induced comparable levels of anti-E. coli antibodies. Thus, the antibody response to the inserted peptide generally parallels the response to the LamB protein (and not to the bulk of E. coli antigens). However, this treatment corresponding to a 'pre-processing' of the recombinant bacteria was not sufficient to obtain an anti-peptide response following s.c. immunization.

Animals↗
PubMed1988

Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria.

A wide variety of peptides in terms of length and sequence can be expressed at the surface of the bacterium Escherichia coli by genetic insertion into a 'permissive' site of the outer membrane protein LamB, used as a carrier. The resulting hybrid proteins essentially keep their biological activities with inserts of up to about 60 amino acid residues, and of a large range of predicted structures or hydrophobicities. This reflects a remarkable flexibility in the organization of the protein, but also in the export machinery. The method used to select such a permissive site is quite general and its potential to generate applications, including a versatile type of live bacterial vaccine, are discussed.

Amino Acid Sequence↗
PubMed1988

Maltose transport and starch binding in phage-resistant point mutants of maltoporin. Functional and topological implications.

The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.

Bacterial Outer Membrane Proteins↗
PubMed1988

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane.

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.

Amino Acid Sequence↗
PubMed1987

Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria.

Having developed a genetic procedure to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier, we apply this procedure to express two distinct portions of the preS2 region of hepatitis B virus: region A, residues 132-145, and region B, residues 153-171. The resulting hybrid proteins (LamB-preS2 A and LamB-preS2 B) were normally expressed, stable, and still kept most biologic functions of LamB. The corresponding bacterial strains were used directly as immunogens in rabbits and mice. Both viral sequences were found to be immunogenic in the two animal species. With LamB-preS2 A, antibodies induced were able to react with the viral particles and the immobilized peptide. With LamB-preS2 B, the antibodies raised were not able to recognize the immobilized peptide. However, the results suggest that the B epitope, inserted in LamB, was at least as efficient as the corresponding synthetic peptide in raising antiviral antibodies. Thus, epitope presentation with LamB may present advantages for immunization. We also have shown that peptide A is an essential part of the polymerized human serum albumin receptor. These results, which validate further the LamB vector system for epitope presentation, provide information on the two hepatitis B regions expressed.

Animals↗
PubMed1987

Bacteriophage lambda receptor site on the Escherichia coli K-12 LamB protein.

We have analyzed eight new phage-resistant missense mutations in lamB. These mutations identify five new amino acid residues essential for phage lambda adsorption. Two mutations at positions 245 and 382 affect residues which were previously identified, but lead to different amino acid changes. Three mutations at residues 163, 164, and 250 enlarge and confirm previously proposed phage receptor sites. Two different mutations at residue 259 and one at 18 alter residues previously suggested as facing the periplasmic face. The mutation at residue 18 implicates for the first time the amino-terminal region of the LamB protein in phage adsorption. The results are discussed in terms of the topology of the LamB protein.

Bacterial Outer Membrane Proteins↗
PubMed1986

malM, a new gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of the malK-lamB operon and encodes a periplasmic protein.

The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied. DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB. The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM. It could encode a protein of 306 amino acid residues. The complete malM open reading frame was cloned under control of the tac 12 promoter. In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3). We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space. We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic. Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.

Amino Acid Sequence↗
PubMed1986

Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.

The LamB protein is a trimeric integral outer membrane protein from Escherichia coli K12 which functions as a pore for maltodextrins and a receptor for bacteriophages. When inserted into two selected sites of LamB, a foreign antigen, the C3 epitope from poliovirus, was exposed at the cell surface with its normal antigenic properties. Since these genetic insertions did not affect in any essential way the routing, activity and folding of the LamB protein, we conclude that the two corresponding LamB sites are at the cell surface as predicted by our recent model. We discuss the implications of our results for the study of protein topology with a single epitope and the direct cloning and cell surface expression of epitopes of interest as well as the development of live vaccines or diagnostic tests.

Amino Acid Sequence↗
PubMed1986

Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12.

Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption. The active form is a trimer. We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins. Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case). The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunodetectable oligomers. Induction of a high level of the four longest of these proteins was toxic to the cell. Five other mutations were due to in-frame insertions. In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction. In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction. Deletions and duplications between some of the inserts were also constructed and studied. To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane. The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers. We tentatively interpret toxicity as due to improper insertion into the outer membrane. Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.

Amino Acid Sequence↗
PubMed1986

[A genetic method for exposing a given epitope at the surface of the bacterium Escherichia coli. Perspectives].

We describe a genetic method leading to insertion of a defined epitope into certain sites of a protein. This method is applied to LamB, an outer membrane protein from Escherichia coli K12. It allowed us to construct an "exposition vector". The bacterial clone harboring this vector with its passenger exposes the epitope at its surface. We discuss briefly some of the perspectives which are opened by this approach concerning the study of structure and localization of proteins, the development of live vaccines and the direct cloning and exposure of unknown epitopes.

Antigens, Bacterial↗
PubMed1985

Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12.

Ten phages which use the LamB protein for adsorption have been isolated from sewage waters. Nine have a shape similar to lambda and require only the LamB protein for adsorption. One has a shape similar to T phages and can use either the LamB or the OmpC protein. Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.

Adsorption↗
PubMed1984

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12.

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

Amino Acid Sequence↗