Escherichia coli is widely used for quick and economic production of heterologous proteins in large quantities. At Genentech we have developed a periplasmic expression platform for antibodies. Typically, heavy chain and light chain of an antibody are each fused to an E. coli secretion signal peptide which mediates the secretion of antibody chains from the cytoplasm to the periplasm. The oxidizing environment and enzymes in the periplasm facilitate correct disulfide bond formation and assembly of heavy and light chains into antibodies. Proteins in E. coli can be secreted to the periplasm through the SecYEG translocon. In this study we investigated the effects of various signal peptides on antibody production. Signal peptides from the heat-stable enterotoxin II (STII), disulfide oxidoreductase (DsbA), maltose-binding protein (MalE), and alkaline phosphatase (PhoA) were fused to the heavy chain of the 5D5 antibody. We found signal peptides significantly affect 5D5 production, with DsbA signal peptide resulting in the highest level of 5D5. Further analysis revealed that the DsbA signal peptide fused to heavy chain resulted in more efficient processing and more periplasmic accumulation of heavy chain, indicating heavy chain is more efficiently translocated to the periplasm when fused to DsbA signal peptide. This talk will focus on the latest results on the mechanism of how signal peptides affect protein secretion to the periplasm and antibody production.

Effects of signal peptides on antibody production in the E. coli periplasm

247th National Meeting (2014)

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