EXPRESSION OF RECOMBINANT ANTI-E-SELECTIN SINGLE-CHAIN FV ANTIBODY FRAGMENTS IN STABLY TRANSFECTED INSECT-CELL LINES

We have investigated the possibility of improving the yield of properl y folded recombinant single chain Fv fragments (sFv) of an antibody by expressing the protein in stably transfected Drosophila melanogaster SC-2 cells. The DNA encoding the variable regions of the 1.2B6 anti-E- selectin antibody were used to generate a recombinant sFv. This constr uct was cloned into the pHEN1 vector for expression in Escherichia col i and the pRmHa-3 vector to generate stably transfected Drosophila SC- 2 cell lines. Following expression in the bacterial system, and using standard refolding protocols to obtain active material, it was shown t hat the majority of the sFv formed non-covalent aggregates, In additio n SDS-PAGE analysis indicated that even the monomeric material was het erogeneous. In contrast, expression of sFv in Drosophila SC-2 cell lin es allowed purification of active sFv directly from the culture supern atant. only a small proportion of the sFv formed aggregates, and the p urified material was homogeneous as determined by SDS-PAGE. Thus the u se of stably transfected insect cells has a number of potential advant ages in expressing recombinant antibody fragments. (C) 1998 Elsevier S cience B.V, All rights reserved.