Modification of the insect cell secretory pathway to enhance posttranslational processing of recombinant proteins.

摘要

The ability to produce high levels of heterologous eukaryotic proteins is the principal reason for the rise in popularity of the baculovirus-insect cell expression system. However, recombinant secreted proteins produced in insect cells are often poorly processed. This deficiency in processing may be due to the occurrence of one or more bottlenecks in the secretory pathway following baculovirus infection. Overexpression of cellular factors believed to be important in protein processing and secretion may be effective in alleviating or eliminating these bottlenecks. Consequently, several cellular processing factors were overexpressed in insect cells in an attempt to enhance production and processing of model secreted proteins.;A secretion bottleneck in cytosol to endoplasmic reticulum polypeptide transport may result from a limitation in cytosolic factors necessary to facilitate the process. To investigate this possibility, a cytosolic heat shock protein 70 (hsp70) was coexpressed in insect cells in concert with immunoglobulin G (IgG). Overexpression of hsp70 increased intracellular soluble and secreted IgG levels. Furthermore, results from pulse-chase labeling experiments suggest that hsp70 enhances IgG processing by increasing the intracellular pool of soluble immunoglobulin precursor light chains.;Although soluble and secreted IgG levels can be increased by overexpression of hsp70, a considerable fraction of immunoglobulin light chain precursor polypeptide produced forms an insoluble aggregate within the cell. This suggests a limitation in signal peptide processing function. In an attempt to overcome this limitation, a bacterial signal peptidase was coexpressed in insect cells with an antibody single chain fragment (scFv). Coexpression of wild type bacterial peptidase with scFv results in a significant enhancement in scFv precursor polypeptide processing.;A sparagine-linked oligosaccharide processing, another posttranslational processing event which may be affected during recombinant protein production, may similarly lend itself to modification by overexpressing enzymes associated with the N-glycosylation processing pathway. Monosaccharide composition and oligosaccharide structural analysis of N-glycans from recombinant transferrin secreted from insect cells overexpressing a beta1-4-galactosyltransferase revealed an increase in galactose-containing oligosaccharides.;The results of this research suggest that modifying the insect cell secretory pathway by overexpression of specific "helper" proteins may be beneficial in enhancing posttranslational processing of certain recombinant secreted proteins.