Genetic engineering strategies for purification of recombinant

Genetic engineering strategies for purification of recombinant antibody fragments
Adi­Bessalem Soniaa, b, Billiald Philippec and Fatima Laraba­Djebaria,b*
Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, BP 32, El –Alia Bab Ezzouar, 16111, Alger, Algérie
b
Laboratoire de Recherche et de Développement sur les Venins, Institut Pasteur d’Algérie
c
Université Paris­Sud, Faculté de Pharmacie & Muséum national d'Histoire naturelle,
F75005 Paris, France
*
Corresponding Author: E­mail: [email protected]/ [email protected]; Fax: (+)213 21 60 33 77
a
ABSTRACT
Genetically engineered antibodies have found many applications in medicine and biotechnology. In this context, various antibody formats have been designed, the most frequently reported of which are single­chain antibody fragments (scFvs). These recombinant antibody fragments can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free or complexed forms with their antigen. The only natural ligands which could recognize antibodies and used as a tool for the affinity purification and detection of scFvs is protein L (PpL). This ligand is a cell wall protein expressed by some strains of the anaerobic species Peptostreptococcus magnus. PpL interacts with the V­ KAPPA framework region 1 (FR1) of some, but not with all, antibody fragments. In this study, polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into an antibody form as a scFv specific to the toxin venom scorpion. After insertion into a vector, scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced scFv presents binding properties similar to those of the original murine antibodies. We also report an easy and efficient method that involves a substitution of consensus sequence for the N­
terminal sequence of the antibody VL domain that does not react with PpL. This substitution did not hinder scFv folding and expression in recombinant bacteria and did not interfere with their antigen­binding function. The preparations of purified scFvs were homogeneous, and no contaminants were detected. In addition, the potent residues able to interact with the PpL were identified by scFv mutant’s construction. The obtained data indicated that the substitution of glutamic acid by glutamine allows scFv to preserve PpL­binding activity, whereas substitution of serine by proline completely eliminates this activity.
This developed technology can be expected to be universal and could provide an efficient strategy for detection and purification of free antibody fragments or coupled fragments to their antigen without required Flag.
Key words: Recombinant antibodies, scFv, Affinity purification, Engineering.