INTRODUCTION
Fn3 3.4.4, a rationally engineered variant of the fibronectin type III (Fn3) domain, plays a pivotal role in extracellular matrix interactions and tumorigenesis regulation. Due to its potential biomedical and therapeutic relevance—especially in cancer-targeting applications—efficient extracellular production of both glycosylated and aglycosylated forms is essential. However, the challenge of secreting functional Fn3 proteins outside the cytoplasmic environment in Escherichia coli has historically hindered scalable production. This study presents a refined strategy that combines genetic engineering, strain selection, and chemical facilitation to achieve high-efficiency extracellular secretion. By utilizing specific signal peptides (SPs) such as OmpA, PelB, and L-AsPsII, along with recombinant expression in strains like E. coli BL21 (DE3) and DKK601, researchers successfully enhanced secretion. The use of Triton X-100 further boosted protein excretion, highlighting its utility as a mild surfactant in extracellular expression systems. Most notably, N-glycosylated Fn3 3.4.4 retained its functionality, binding effectively to mesothelin (MSLN) as verified by ELISA. This work not only advances understanding of extracellular protein production but also supports industrial-scale development of therapeutic proteins.
ENGINEERED SIGNAL PEPTIDES FOR EFFICIENT EXCRETION
Signal peptides (SPs) are critical determinants of successful protein localization and secretion in bacterial systems. In this study, three SPs—OmpA, PelB, and L-AsPsII—were employed to direct the transport of Fn3 3.4.4 to the periplasm or extracellular medium. Among these, PelB was particularly efficient in delivering the protein to the periplasm due to its superior cleavage rate, allowing the accumulation of soluble, correctly folded Fn3. Conversely, OmpA showed promise in directing N-glycosylated Fn3 3.4.4 out into the culture medium, highlighting its potential for extracellular expression. The strategic comparison of SP performance demonstrated that proper selection and pairing of SPs with target proteins is crucial for maximizing expression and downstream recovery. This insight is valuable for optimizing secretion of other glycoproteins in bacterial platforms.
N-GLYCOSYLATION AND FUNCTIONAL PROTEIN EXPRESSION
N-glycosylation is often essential for protein folding, stability, and bioactivity, yet it remains a complex task in E. coli due to its lack of native glycosylation pathways. This study leveraged engineered systems to express N-glycosylated Fn3 3.4.4 in E. coli, resulting in successful secretion into the culture medium, particularly under the guidance of the OmpA signal peptide. Importantly, the glycosylated protein maintained its affinity to mesothelin (MSLN), confirming structural integrity and biofunctionality. These findings reinforce that bacterial expression systems, when correctly tuned, can serve as viable hosts for glycoprotein production—a key advancement for recombinant therapeutics.
STRAIN SELECTION: BL21 (DE3) VS DKK601
Selecting the right bacterial host is pivotal for efficient recombinant protein expression. The study evaluated two commonly used strains: E. coli BL21 (DE3), known for high-level protein expression, and DKK601, an engineered strain optimized for glycosylation compatibility. While BL21 (DE3) showed robust performance in expressing aglycosylated Fn3 3.4.4, DKK601 demonstrated superiority in supporting N-glycosylation machinery. This differential performance underscores the importance of aligning host strain capabilities with the biochemical requirements of the target protein. It also highlights DKK601 as a promising chassis for future glycoengineering applications.
TRITON X-100 ENHANCES EXTRACELLULAR PROTEIN RELEASE
Chemical additives like Triton X-100 can significantly influence protein excretion without compromising cellular viability. In this study, low concentrations of Triton X-100 were used post-induction to facilitate the release of Fn3 3.4.4, particularly the N-glycosylated form, into the culture medium. Acting as a mild non-ionic surfactant, Triton X-100 likely disrupted membrane tension, enhancing leakage or secretion pathways. This approach presents a cost-effective, scalable solution for extracellular production and purification, particularly when paired with optimal signal peptides and host strains. These findings pave the way for simplified downstream processing in protein manufacturing pipelines.
APPLICATIONS IN BIOTHERAPEUTIC DEVELOPMENT
The demonstrated extracellular expression and functional validation of N-glycosylated Fn3 3.4.4 open promising avenues in therapeutic development. Fn3 domains are widely explored for their small size, stability, and targeting potential—especially in cancer therapies involving antigens like mesothelin. The ability to produce functional glycosylated Fn3 variants in E. coli enables faster, more economical production compared to eukaryotic systems. Moreover, this scalable platform can be extended to engineer and produce other glycoprotein-based biotherapeutics, making it a valuable tool for biotechnology, diagnostics, and personalized medicine.
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