Biomedical subjects

F Breitling

Publications and source records attributed to F Breitling.

7 recordsLinked to original sources

PubMed1992

A vector for the removal of deletion mutants from antibody libraries.

To reduce the number of deletion mutants from antibody (Ab) libraries that had been amplified by PCR from peripheral blood lymphocytes, we constructed the Ab expression vector, pLAB, in which DNA coding for a single-chain Ab was inserted into the gene encoding beta-lactamase (Bla) at the 3'-terminus of its signal sequence. After transforming Escherichia coli with this vector, a fusion protein with a functional Bla domain was produced that was able to protect the bacteria from the action of ampicillin (Ap). Libraries can therefore be usefully propagated with this vector, since only those clones carrying inserts that are in frame with Bla will survive Ap selection, while others that carry out-of-frame deletions or internal stop codons are eliminated.

Amino Acid Sequence↗

PubMed1991

A surface expression vector for antibody screening.

To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili. Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG. Ab::pIII was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences. It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen. The phagemid DNA is even smaller than that of wt fd phages and can easily be propagated in plasmid form. Most importantly, its Ab::pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants. After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.

Amino Acid Sequence↗

PubMed1991

Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein.

To target recombinant antibodies to the surface of Escherichia coli, we have fused single-chain variable domains to its peptidoglycan associated lipoprotein (PAL). The fusion protein was able to bind antigen and was tightly bound to the murein layer of the cell envelope. Antibody-PAL had little effect on cell growth and viability. In contrast, the expression of single chain antibody alone eventually resulted in cell lysis. Immunofluorescence studies on unfixed cells showed that functional antibodies were accessible at the surface of intact bacteria. This could provide a means of isolating single cells producing specific antibodies from libraries in E. coli by fluorescence assisted cell sorting (FACS). Pal fusions may also be of general interest for the presentation of proteins at the surface of E. coli as, for example, in the production of live vaccines.

Amino Acid Sequence↗

PubMed1987

Location of the epitope for the alpha-tubulin monoclonal antibody TU-O1.

A cyanogen bromide peptide of pig brain alpha-tubulin with high reactivity to the monoclonal antibody TU-O1 has been isolated and identified. It corresponds to positions 37-154 of the alpha-tubulin sequence. A tryptic peptide within this region corresponding to positions 65-79 was also immunoreactive. Its relatively low reactivity, however, indicates that one or more important determinants are missing.

Amino Acid Sequence↗

PubMed1986

Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B.

Tryptic and cyanogen bromide peptides of pig brain alpha- and beta-tubulin reacting with monoclonal antibodies YOL1/34, DM1A and DM1B have been isolated and identified. They all correspond to parts of the C-terminal regions of either alpha- or beta-tubulin, and those peptides reacting with a given antibody have overlapping sequences. In the case of YOL1/34, its relatively high reactivity with small peptides suggests that many of the determinants for this antibody are within the overlapping region of these peptides comprising only nine amino acids in positions alpha 414 to 422. The smallest common region of peptides reacting with the other alpha-tubulin antibody DM1A corresponds to positions alpha 426 to 450, whereby amino acids within the positions 426 and 430 appear to be particularly important for reactivity. Since the last C-terminal residues of alpha-tubulin are also accessible to antibodies and enzymes, it seems that an extensive part (35 to 40 residues) of this very acidic C-terminal domain is exposed on the surface of native tubulin dimers. In microtubules, however, the amino-terminal end of this region appears to be less accessible, as YOL1/34 reacts poorly, if at all, with intact microtubules. All of the peptides reacting with beta-tubulin monoclonal antibody DM1B were derived from the acidic C-terminal domain and they overlapped in positions beta 416 to 430. This indicates that beta-tubulin is also positioned with at least part of its acidic C-terminal domain on the surface of microtubules, since DM1B reacts with unfixed microtubules after microinjection.

Amino Acid Sequence↗

PubMedDate not supplied

Regulated secretion and purification of recombinant antibodies in E. coli.

A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.

Amino Acid Sequence↗

PubMedDate not supplied

Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.

Antibodies, Monoclonal↗