Ask the Expert: A Novel Escherichia coli System for Controlled Release of Difficult-to-Secrete Proteins

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Although Escherichia coli often enables dependable, highly productive expression of nonglycosylated recombinant proteins, the efficiency with which it secretes a target protein into culture supernatant can depend greatly on that molecule’s physicochemical properties. Some proteins remain trapped in periplasm, thus diminishing process yield and productivity. In June 2021, Marcel Thoen (head of Wacker Biotech’s Global Competence Center for Cell Line Development) described how his company’s improved ESETEC (E. coli secretion technology) solutions can address productivity challenges raised by difficult-to-secrete recombinant proteins.

Thoen’s Presentation
Thoen emphasized that improving E. coli’s periplasmic-secretion efficiency raises clear advantages for upstream processing. Doing so can increase yields significantly, especially when compared with titers achieved with conventional microbial systems that generate soluble proteins. Enhancing a host’s secretion efficiency also could eliminate the need for homogenization, inclusion body (IB) preparation, and protein refolding, enabling scientists to usher fermentation supernatant into primary purification steps. Together, such benefits could reduce cost of goods (CoG) by five fold as compared with expenses incurred by traditional microbial-expression processes.

ESETEC periplasmic-expression systems leverage a well-characterized E. coli K12 derivative that can be induced by a Tac promoter and isopropyl β-D-1-thiogalactopyranoside (IPTG) to secrete target proteins into culture broth. Genes of interest (GoIs) are delivered to an ESETEC host using any of several pEX-based plasmids that yield high or low copy numbers depending on customer needs. The plasmids are designed to guide translocation of target proteins using proprietary signal sequences.

However, some proteins are difficult for host periplasm to release. Thoen presented a case study describing production of a Wacker customer’s antibody fragment (Fab). Using a conventional ESETEC system, Wacker generated 4 g/L of product, but only 0.5 g/L of properly folded material could be obtained from the resulting supernatant. Wacker later determined that uncontrolled retention time — and thus unintended interactions among targets, chaperones, and folding factors — allowed secretion of improperly folded material.

Improved ESETEC systems address that problem by applying a “controlled secretion” principle that tailors a protein’s periplasm retention time. The process effectively decouples a host’s protein production and release phases, Thoen explained. IPTG initiates product generation and accumulation in the periplasm; protein secretion is triggered subsequently by addition of a second inducer that stimulates formation of a distinct release factor. By controlling how long proteins remain in the periplasm, Thoen continued, improved ESETEC systems also enhance management of their interactions with chaperones, isomerases, and other folding factors, helping to ensure correct protein structure and thereby increasing process robustness.

Referring back to his case study, Thoen reported that the improved ESETEC system secreted 4 g/L of the customer’s Fab, equating to an eight-fold improvement in titer from that of the conventional ESETEC system. Subsequent testing by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the new strain secreted the Fab’s light and heavy chains in equimolar amounts. Supernatant obtained from the process also showed low viscosity, raising distinct advantages for primary filtration and purification steps.

To determine the impact of controlled secretion on supernatant quality, Wacker also evaluated host-cell protein (HCP) concentrations using Western blot analyses. The company focused especially on thermounstable elongation factor (EF-Tu), a cytoplasmic impurity endemic to prokaryotes. Proteins representative of EF-Tu could not be identified in samples from either ESETEC strain. However, compared with the conventional system, the improved ESETEC strain showed 67% fewer total HCPs and 71% less endotoxin per milligram of secreted Fab. Such results, Thoen explained, show that controlling secretion and release can improve supernatant quality significantly.

Questions and Answers
What secretory titers can the improved ESETEC system achieve? Although production levels differ across protein products, the system generally increases titers for hard-to-secrete nonglycosylated proteins by 3–8×. Record titers for Fabs and enzymes are ~7 g/L and ~15 g/L, respectively.

How does the ESETEC system ensure proper protein folding? Wacker can apply any of several proprietary chaperone proteins and isomerases to guide that process. The company can consult with customers during feasibility study to ensure appropriate selection of such tools.

Watch the complete presentation online.