Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen (original) (raw)
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Journal of Biological Chemistry, 1991
Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella 0-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. V, and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were-50 mglliter, chiefly in the form of inclusion bodies. With VL in the NH2terminal position, the amount of secreted product obtained was 2.4 mglliter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG. The antigen-binding site of an antibody is formed by the non-covalent association of the variable domains (VL1 and VH) at the amino termini of the heavy and light chains. For in uiuo diagnostic and immunotherepeutic applications, small antibody fragments are desirable because of reduced immunogenicity and shorter tissue clearance times (31). Fv fragments consisting of the VL and VH domains alone are such molecules, but attempts to isolate Fv fragments by proteolytic
Strategies for the expression of antibody fragments in Escherichia coli
This article summarizes the techniques for the expression of various antibody fragments in Escherichia coli. The properties of various antigen binding fragments are discussed and several strategies for expression are compared. Emphasis is placed on the secretory approach, as it leads directly to functional fragments and thus forms the basis for all screening approaches, be it with cells or phages. Purification procedures of the fragments from E. coli are also discussed.
Proceedings of The National Academy of Sciences, 1995
A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level
Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli
Proceedings of the National Academy of Sciences, 1984
Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.
Vaccine, 1994
Salmonella strains have great potential as live carriers of heterologous antigens to induce immunity against a variety of infectious diseases. However, the amount of heterologous antigen required to induce an adequate immune response may be toxic.for the bacterium and result in cell death, overattenuation or loss of expression of the heterologous antigen. To solve this problem an expression vector was developed with a strong promoter located on a DNA fragment which is inverted at random. Antigen is only expressed in one particular orientation of the promoter. Thus a bacterial population harbouring the plasmid will consist of a subpopulation which does not produce heterologous antigen, and is therefore not affected in growth, persistence and dissemination within the host. Further, this non-producing population will continuously segregate antigen-producing bacteria. To evaluate the system, CtxB was used as a model antigen. Analysis of the plasmid DNA isolated from Salmonella revealed a selection against the promoter orientation that directs transcription of the ctxB gene. In spite of this', the vector was stably maintained in vivo and induced CtxB-specific IgA and IgG in mice. These results indicate that this kind of expression vector may offer a solution to the problem of unstable expression of foreign antigens in live bacterial vaccine strains.
Expressing antibodies in Escherichia coli
There are many reasons why researchers may wish to work with recombinant antibodies: for answering basic questions of protein architecture, specific binding and protein folding, for applying the modified antibodies in biology and biotechnology (ranging from biosensors to affinity chromatography), or for use in medicine. At the beginning of each project there is the problem of producing the protein in sufficient quantities. This problem will still be encountered, even if the antibody is first selected from a phage library. This chapter will summarize methodology for cloning, expression, purification, and detection of recombinant antibodies, using Escherichia coli, the most familiar host in biochemical laboratories.