Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen

Anand, N.N.; Dubuc, G.; Phipps, J.; MacKenzie, Colin R.; Sadowska, Joanna; Young, N. Martin; Bundle, David R.; Narang, Saran A.

doi:10.1016/0378-1119(91)90347-e

Cited by 34 publications

(9 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have isolated a monoclonal antibody Fab, designated CCRC-R1, that specifically binds the plant cell wall polysaccharide RG-II. Although several anti-polysaccharide hybridomas have been molecularly cloned and expressed (Mainhart et al, 1984;Anand et al, 1991;Adderson et al, 1993), this is a nove1 use of phage-display libraries or recombinant methods for de novo isolation of an antibody against any plant antigen or against any polysaccharide. This is also the first antibody we have raised against RG-II.…”

Section: Discussionmentioning

confidence: 99%

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

et al. 1996

View full text Add to dashboard Cite

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.

show abstract

Section: Discussionmentioning

confidence: 99%

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

et al. 1996

View full text Add to dashboard Cite

show abstract

“…Protein expression was induced by adding isopropyl‐β‐ d ‐thiogalactopyranoside (1 m m ). After induction, the culture was shaken for an additional 72 h at 25°C, and the periplasmic fraction was extracted by the osmotic shock method (Anand et al . 1991).…”

Section: Methodsmentioning

confidence: 99%

The blood–brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Abulrob

Sprong

Henegouwen

et al. 2005

Journal of Neurochemistry

176

128

View full text Add to dashboard Cite

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptormediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K + depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-bcyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, a(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes a(2,3)-, but not with Sambucus nigra agglutinin that recognizes a(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal a(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.

show abstract

“…Fab Gene Construction. The dicistronic Fab construct pSal-Fab (Anand et al, 1991a) consisting of the Fd and light chain genes, each preceded by the ompA signal peptide, in the expression plasmid pUCE8 (Narangetal., 1987) was modified somewhat prior to the initiation of this study. An unpaired cysteine (Cys94L) was mutated to Ser, using the codon TCT, to eliminate unwanted disulfide bond formation.…”

Section: Methodsmentioning

confidence: 99%

“…As a model system for this type of study, the Salmonella antibody offers several advantages in addition to the well-refined crystal structure. These include a detailed description of binding thermodynamics (Sigurskjold et al, 1991; Sigurskjold & Bundle, 1992) and an efficient Escherichia coli expression system (Anand et al, 1990(Anand et al, , 1991a that yields active product through the use of bacterial signal peptides (Better et al, 1988;Skerra & Pluckthun, 1988). With a binding constant of 2 X 105 M_1, the anti-Salmonella antibody has very good affinity for an anti-carbohydrate antibody.…”

mentioning

confidence: 99%

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: Role of the heavy-chain CDR3 residues

Vp²,

Nn³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

show abstract

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen

Cited by 34 publications

References 11 publications

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

The blood–brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: Role of the heavy-chain CDR3 residues

Contact Info

Product

Resources

About