Bacterial Expression and Purification of the Fab Fragment of a Monoclonal Antibody Specific for the Low-Density Lipoprotein Receptor-Binding Site of Human Apolipoprotein E

Raffaı̈, Robert L.; Vukmirica, Jelena; Weisgraber, Karl H.; Rassart, Éric; Innerarity, Thomas L.; Milne, Ross W.

doi:10.1006/prep.1999.1031

Cited by 12 publications

(9 citation statements)

References 15 publications

(15 reference statements)

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“…Two overlapping mutant products were created by performing PCR with forward and reverse mutant oligonucleotides in conjunction with a 3Ј-reverse primer containing the SpeI restriction site and a 5Ј-forward primer containing the XhoI site. The spliced overlap mutant PCR product was gel-purified, digested with the two endonucleases, and subcloned into the pComb3 soluble expression vector as described previously (18). In all cases, the amplification conditions consisted of 30 repeated cycles on a Stratagene Robocycler PCR apparatus.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant Fab Production and Purification-The production and purification of the 2E8 recombinant Fab (rFab) fragment have been described (18). Each rFab preparation was subjected to SDS-PAGE, resolved by isoelectric focusing PAGE (Bio-Rad) in parallel with the pure hybridoma-generated Fab fragment, and stained with Coomassie Blue.…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme-linked Immunosorbent Assay (ELISA) of 2E8 rFab Fragments-The binding of 2E8 variants to immobilized apoE was measured by an indirect ELISA as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

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Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces

Raffaı̈¹,

Weisgraber²,

MacKenzie³

et al. 2000

Journal of Biological Chemistry

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Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/ apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. 57 3 Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8⅐apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.ApoE, a 34-kDa protein composed of 299 amino acids, is an important functional component of chylomicrons and very low, intermediate, and high density lipoproteins. As a ligand for the low density lipoprotein receptor (LDLR) 1 and for other members of the LDLR family, it plays an important role in the metabolism of plasma lipoproteins. ApoE exists as three common isoforms: apoE2, apoE3, and apoE4. The most common isoform, apoE3, has cysteine and arginine at positions 112 and 158, respectively, whereas apoE2 has cysteine and apoE4 has arginine at both positions. ApoE2 binds poorly to the LDLR, and homozygous inheritance of the apoE2 allele is strongly associated with type III dyslipoproteinemia (1).Several lines of evidence suggest that positively charged amino acids at positions 136 -150 of apoE interact directly with the LDLR (2). The LDLR-binding site is in the 22-kDa aminoterminal domain, and the strongest lipid-binding region is in the 10-kDa carboxyl-terminal domain. In a lipid-free form, the 22-kDa amino-terminal domain is folded into an elongated four-helix bundle; basic residues that have been implicated in LDLR binding are situated on helix 4, where they form a region with strong electropositive potential (3). Arg 158 does not participate directly in apoE-mediated binding to the LDLR, but its replacement by a cysteine in apoE2 causes the rearrangement of intramolecular salt bridges within the LDLR-binding site, altering the alignment of the positively charged residues of apoE that interact with the LDLR (4).The LDLR, an integral membrane protein composed of 831 amino acids, mediates the uptake of lipoproteins through its ability to bind to apoE and apoB. The amino terminus of the LDLR contains the ligand-binding domain that is composed of seven imperfect repeats of 40 amino acids. Each repeat includes six conserved cysteine residues and a cluster of acidic resi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces

Raffaı̈¹,

Weisgraber²,

MacKenzie³

et al. 2000

Journal of Biological Chemistry

Self Cite

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“…Currently, no rationale has been established for the different expression profiles observed in different E. coli strains. Raffai et al (1999) previously reported that TG1 cells are more suitable for expressing 2E8 Fab than XL1-Blue, and suggested that this difference is due to different bacterial growth rates. Raffai et al (1999) also observed a higher yield of Fab within the periplasm and suggested that the relative distribution of antibody fragments between the periplasmic space and the medium can be modulated by culture conditions, as was also proposed by Kipriyanov et al (1997).…”

Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan¹,

Ong²,

Nathan³

2004

BMB Reports

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A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

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“…The human constant domains confer established and standardized detection and purification means of Fab derived from multiple species as well as improving the E. coli expression level of Fab. 16,17) Therapeutically, chimeric mouse/human Fab can readily be channeled into previously reported strategies for complete humanization. 14,18) The phage library displaying chimeric mouse/human Fab was panned against immobilized B. pseudomallei protease.…”

Section: Resultsmentioning

confidence: 99%

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

Nathan

Rader

Barbas

2005

Bioscience, Biotechnology, and Biochemistry

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Bacterial Expression and Purification of the Fab Fragment of a Monoclonal Antibody Specific for the Low-Density Lipoprotein Receptor-Binding Site of Human Apolipoprotein E

Cited by 12 publications

References 15 publications

Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces

Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

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