Type III secretion as a generalizable strategy for the production of full‐length biopolymer‐forming proteins

Abstract

Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its applicability to an array of engineered biomaterial-forming peptides lend support for the application of bacterial expression and secretion for other proteins that are traditionally difficult to express and isolate from the bacterial cytoplasm. Biotechnol. Bioeng. 2016;113: 2313-2320. © 2015 Wiley Periodicals, Inc.

Type III secretion systems: the bacterial flagellum and the injectisome

“…Proteins of choice can easily be targeted to the machinery by a short N-terminal signal (which may be cleaved off at a later stage), and are specifically exported into the supernatant, which leads to significant enrichment of soluble protein without additional purification steps. Both types of T3SS have been used for this purpose: the injectisome was used to export various proteins, including spider silk proteins [261], and has recently been shown to be a useful tool for the production of biopolymer-forming proteins, with increased export levels upon overexpression of the master transcriptional regulator [262]. Similarly, the flagellar T3SS was shown to be able to export a variety of different protein types [191] and subsequently optimized for protein secretion [263].…”

Proteins adopt functionally active conformations after type III secretion

“…Production of heterologous proteins via secretion to the extracellular space holds many advantages over intracellular accumulation: purification is simplified; cytotoxicity is alleviated; and cell lysis is not required [1, 2]. The production and secretion of heterologous proteins can be achieved using the T3SS of various Gram-negative bacteria [7–9, 26, 27]. However, the mechanism of protein secretion requires an unfolding event during translocation [5].…”

An estimate is worth about a thousand experiments: using order-of-magnitude estimates to identify cellular engineering targets

“…By introducing transcriptional control, we manipulate these two key variables simultaneously and achieve a ~ tenfold increase in secreted protein titer [34]. Engineering improvements that were identified by rough estimates resulted in a bacterial strain that was able to produce and secrete heterologous proteins at high titer and enabled the production of difficult-to-express repetitive proteins [35]. We expect increased product titer by further manipulation of the five aforementioned variables.…”