Cloning, expression and efficient refolding of carbohydrate-peptide mimicry recognizing single chain antibody 2D10.
Carbohydrate-peptide mimicry was found to be manifested through the cross-reactivity of an anti-mannopyranoside monoclonal antibody 2D10 (mAb-2D10) with YPY motif containing 12-mer peptide (DVFYPYPYASGS). Such multiple binding options for a monoclonal antibody could emanate from the possible flexibility of the antigen combining site. To address the molecular details of this phenomenon, single chain antibody (scFv) containing the antigen combining variable domain of mAb-2D10 was constructed. The present work describes the cloning, expression, purification and efficient refolding of scFv-2D10 and its His(6) tag fusion variants. The scFv expressed poorly in soluble/active form in the periplasmic compartment and concurrently exhibited higher tendency towards accumulation in inclusion bodies inside the Escherichia coli cytoplasm. The scFv was refolded from the inclusion bodies with approximately 68% yield using a previously described protocol which employed concomitant removal of the chaotropic and oxidizing reagents along with the additives. However, their differential removal, as described in the present report resulted in approximately 97% effective yield of the soluble scFv-2D10, an increase of 42%. The binding kinetics of the refolded scFv for both the mimicking ligands was examined using surface plasmon resonance experiments. The scFv-2D10 exhibited binding affinities similar to those reported for mAb-2D10 (IgG) showing that the modifications introduced in the refolding protocol have facilitated efficient preparation of active 2D10 scFv.
Tapryal
Suman
S
National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India.
Krishnan
Lavanya
L
Batra
Janendra K
JK
Kaur
Kanwal J
KJ
Salunke
Dinakar M
DM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008358:Mannose / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D010455:Peptides; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D008358:Mannose