Engineering Secretion Machinery for High-Throughput Protein Production

Protein production traditionally relies on cytosolic accumulation, such that extensive purification steps are required to separate the desired product from the intracellular milieu. During protein engineering experiments, it is often desirable to characterize many variants, and the limited throughput of production by cytosolic accumulation therefore limits the number of variants that can be examined. We envision using secretion to enable high-throughput protein production and analysis of libraries of proteins. Secretion offers the benefits of simplified purification steps, but low titers have limited the applicability of this strategy, particularly in bacterial hosts. To this end, we recently engineered a type III secretion system to achieve as much as a 100-fold increase in secretion titer of heterologous proteins, giving rise to titers of >100 mg/L of protein in the extracellular space. We applied the engineered system to the production of various classes of proteins, including enzymes, biomaterial-forming proteins [1], and antimicrobial peptides. After a single purification step, the secreted proteins were >90% pure as compared to 35-65% purity from cytosolic purification. Moreover, after secretion the enzymes were active and the biopolymer-forming proteins could be cross-linked to form materials with highly reproducible and tunable properties. We note that a few of the heterologous proteins tested were incompatible with secretion; we are currently establishing the key parameters that govern “secretability” via the type III machinery. In this presentation, I will describe the three-pronged strategy used to improve secretion titers: 1) gaining control over secretion machinery regulation [2], 2) engineering the needle-forming protein, and 3) adding components to the media. I will also discuss our development of a model that uses the primary sequence to predict which heterologous proteins will be secreted, and detail how we applied this enhanced system to develop antimicrobial hydrogels for tissue culture applications.

 

1. Azam A., Metcalf K..J., Li C., Tullman-Ercek D. “Type III Secretion as a Generalizable Strategy for the Development of Peptide-based Materials.” (Submitted).

 

2. Metcalf K.J., Finnerty C., Azam A., Valdivia E., Tullman-Ercek D. (2014) “Using transcriptional control to increase titer of secreted heterologous proteins by the type III secretion system.” Appl. Environ. Microbiol. (In press).