Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti-HIV mAb 4E10 from transgenic tobacco.
Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.
Platis
Dimitris
D
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.
Maltezos
Anastasios
A
Ma
Julian K-C
JK
Labrou
Nikolaos E
NE
D000327:Adsorption; D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D032701:Biomimetics; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics*
D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands