Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Abstract

A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…In fact, multiple reactions within the biosynthetic pathway of MAb seem amenable for improvement (O'Callaghan et al, 2010). This notion is supported by non-limiting examples of studies demonstrating an increase in volumetric productivity or q p in CHO cells upon the expression of effector genes related to translation and translocation (Le Fourn et al, 2014), ER folding processes (Borth et al, 2005;Chung et al, 2004;Hwang et al, 2003) and protein transport and secretion (Florin et al, 2009;Peng et al, 2011;Peng and Fussenegger, 2009) (Fig. 1).…”

“…1). Several reports have demonstrated synergistic effects on the volumetric productivity or q p of co-expressing multiple genes in CHO cells (Cain et al, 2013;Le Fourn et al, 2014;Mohan and Lee, 2010;Peng and Fussenegger, 2009), demonstrating the potential of combining effector genes. In addition to effector gene overexpression, downregulating endogenous genes might be equally important for increasing q p (Feichtinger et al, 2016;Harreither et al, 2015).…”

Organization of SNAREs within the Golgi Stack

“…Thus, it appears that SM proteins can control the amount of different Q a -SNAREs. Remarkably, the overexpression of SM proteins can boost protein secretion by increasing the size of secretory organelles in certain cells (Peng and Fussenegger 2009). …”

Mammalian cells as biopharmaceutical production hosts in the age of omics

“…3). Although no overexpression studies for targets identified using omics data have yet been reported, Peng et al [64] overexpressed two SNARE proteins involved in vesicle trafficking: Sly1 and Munc18c. This strategy lead to an increased q of Rituximab of up to 15-fold in CHO cells [64].…”