Comparison of three microbial hosts for the expression of an active catalytic scFv.
Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.
Robin
Sylvain
S
FRE-CNRS no. 2230 Biocatalyse, Faculté des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 03, France.
Petrov
Kliment
K
Dintinger
Thierry
T
Kujumdzieva
Anna
A
Tellier
Charles
C
Dion
Michel
M
D000818:Animals; D017151:Antibodies, Catalytic / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006241:Haptens / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D007716:Kluyveromyces / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D017151:Antibodies, Catalytic; D006241:Haptens; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004247:DNA