Modifications of a signal sequence for antibody secretion from insect cells

Abstract

Monoclonal antibodies and antibody fragments have recently been developed for use in diverse diagnostic and therapeutic applications. Insect cells can efficiently secrete recombinant proteins such as antibody molecules through post-translational processing and modifications that are similar to those performed in mammalian cells. In eukaryotic cells, the signal sequence in a nascent polypeptide is recognized by the signal recognition particle, and the polypeptide is then folded and modified in the endoplasmic reticulum. The signal sequence consists of three regions, a positively charged N-terminus, a hydrophobic core, and a polar C-terminus. In the present study, we examined the substitutions of the characteristic amino acids of a Drosophila immunoglobulin heavy chain binding protein signal sequence, and investigated the effect on the secretory production of an antibody Fab fragment from lepidopteran insect cells in transient expression. A modification of the signal sequence for the heavy chain resulted in a twofold increase in the secreted Fab fragment, while the modification for the light chain led to a more than 3.6-fold increase.

Keywords: Antibody; Fab fragment; Insect cell; Recombinant protein production; Secretory production; Signal sequence.

Fig. 1

Improved secretion of an Fab fragment by using a modified BiP signal sequence upstream of the heavy chain (Hc) gene. a Antigen-binding activity of an Fab fragment, as detected by enzyme-linked immunosorbent assay (ELISA). The binding activity obtained with the native BiP signal sequence was indicated as 100%. Error bar = 1 SD (n = 3). b Fab fragment (Mw 50,300), as detected by western blotting

Fig. 2

Improved secretion of an Fab fragment via a modified BiP signal sequence upstream of the light chain (Lc) gene. a Antigen-binding activity of an Fab fragment, as detected by ELISA. The binding activity obtained with the native BiP signal sequence was indicated as 100%. Error bar = 1 SD (n = 3). b Fab fragment (Mw 50,300), as detected by western blotting

Fig. 3

Effect of double modifications in the BiP signal sequence upstream of the Lc gene. a Antigen-binding activity of an Fab fragment, as detected by ELISA. Error bar = 1 SD (n = 3). b Fab fragment (Mw 50,300), as detected by western blotting

Fig. 4

Effect of the simultaneous use of Hss12 and Lss12. a Antigen-binding activity of an Fab fragment, as detected by ELISA. Error bar = 1 SD (n = 3). b Fab fragment (Mw 50,300), as detected by western blotting