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 properly 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 construct was cloned into the pHEN1 vector for expression in Escherichia coli 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 that the majority of the sFv formed non-covalent aggregates. In addition SDS-PAGE analysis indicated that even the monomeric material was heterogeneous. In contrast, expression of sFv in Drosophila SC-2 cell lines allowed purification of active sFv directly from the culture supernatant. Only a small proportion of the sFv formed aggregates, and the purified material was homogeneous as determined by SDS-PAGE. Thus the use of stably transfected insect cells has a number of potential advantages in expressing recombinant antibody fragments.
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
D L
DL
Mahiouz
G
G
Aichinger
D O
DO
Haskard
A J
AJ
George
D000818:Animals; D000918:Antibody Specificity; D015374:Biosensing Techniques; D003001:Cloning, Molecular / Q000379:methods; D004331:Drosophila melanogaster / Q000166:cytology; D019040:E-Selectin / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D019040:E-Selectin; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv