The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key target in COVID-19 vaccine development, but its complex structure poses expression challenges. Here, we show how cell-free protein expression using ALiCE® enabled rapid expression of RBD with accurate post-translational modifications, delivering functional, immunogenic antigens. By eliminating the limitations of cell-based systems, ALiCE® has the potential to enable timely and scalable antigen production, accelerating vaccine development pipelines.
Antigens – A Bottleneck in Vaccine Development
Identifying antigenic sequences and validating their immunogenicity are vital first steps in the development of any vaccine. Yet, the expression of antigenic proteins is often a limiting factor in vaccine development, particularly those that are difficult to express in commonly used host expression systems.
This challenge is especially pronounced for protein-based vaccines. Known for their stability and reduced cold chain requirements, protein-based vaccines offer distinct advantages over newer modalities.1 Yet, they are not the go-to modality for rapid response to emerging epidemic threats, as successful expression of high-quality antigens and candidates is painfully slow and time-consuming. Traditional cell-based systems, such as mammalian, yeast, or insect cells, often require time-intensive optimization of expression conditions, causing significant delays.
Bottlenecks in expression are not limited to early development. Scaling up production to support preclinical studies, clinical trials, and subsequent manufacturing is also slow and challenging. Reliable expression platforms capable of producing vaccine-related proteins in a timely manner are key to the success of vaccine development.
The Protein in Profile: SARS-CoV-2 Spike Protein Receptor-Binding Domain
The RBD of the SARS-CoV-2 spike protein is central to the virus’s ability to infect host cells, making it a key target in vaccine and therapeutic research. This domain interacts directly with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells, which facilitates viral entry and subsequent infection. Its key role in infection and high conservation across SARS-CoV-2 variants makes it an ideal immunogen for COVID-19 vaccine development.2
The significance of the RBD extends beyond its functional role in viral entry. Structurally, the RBD is a complex 25 kDa monomer characterized by four disulfide bonds and two N-glycosylation sites. The specific arrangement of glycosylation sites and disulfide bonds influences how the immune system recognizes the protein.3 This makes the RBD a benchmark for assessing the capabilities of expression platforms in producing immunogens with native-like properties.
Streamlining Production Through Cell-Free Expression
Cell-free protein expression presents a powerful alternative to traditional cell-based antigen expression systems, removing the need for living cells and accelerating production. This method is particularly beneficial for rapid responses to emerging pathogens, as it allows for quick optimization of expression conditions. Once the ideal expression conditions are determined, scalable production of antigens is also possible with cell-free expression systems to meet diverse research and manufacturing needs.
In addition to rapid optimization and scale-up capabilities, eukaryotic cell-free platforms can support the production of proteins with complex PTMs. For example, ALiCE® can produce proteins within microsomal structures that mimic the endoplasmic reticulum, facilitating proper glycosylation and disulfide bond formation. The ability to incorporate PTMs precisely is critical for the functionality and immunogenicity of antigens like the SARS-CoV-2 RBD.
In comparison to cell-based methods, cell-free protein production also lowers contamination risks and provides greater control over reaction conditions. By removing the limitations of cell viability and proliferation, cell-free platforms improve the production of complex antigens, making them ideal tools for vaccine development and pandemic preparedness.
Materials and Methods
To produce the full-sized SARS-CoV-2 spike protein and the RBD, both constructs were cloned into the pALiCE02 vector. This vector directs expressed proteins to microsomes which serve as a scaffold for membrane proteins and mediate essential PTMs such as glycosylation and disulfide bond formation, ensuring proper folding and immunogenicity of the RBD.
Purification: The proteins were subsequently purified using Strep-tag II affinity purification with Strep-Tactin XT 4Flow resin (IBA Lifesciences).
Tagging: A HaloTag was incorporated at various C-terminal positions in the constructs to identify the most effective position. This tag was selected for its utility in downstream purification and immobilization applications. Additionally, a Strep-tag II was included in the constructs to facilitate protein purification.
Expression and microsomal disruption: The ALiCE cell-free expression reaction was run in a 10 mL volume for 48 hours. To release expressed proteins, microsomal disruption was performed using DDM, followed by fractionation as per the ALiCE® Instruction Manual.
KEY OPTIMZATION STRATEGY
Following microsomal disruption, we chose fractionation instead of the faster one-step protocol available in the ALiCE user manual. We recommend fractionation to obtain ultrapure proteins suitable for downstream applications, such as binding assays.
RESULTS
Successful Production and Purification of SARS-CoV-2 RBD
Following microsomal disruption, we chose fractionation instead of the faster one-step protocol available in the ALiCE user manual. We recommend fractionation to obtain ultrapure proteins suitable for downstream applications, such as binding assays.
All constructs were successfully expressed in the ALiCE® cell-free reaction. Following microsomal disruption and purification with Strep-Tactin XT 4Flow resin, a single, strong band was visible for the 25 kDa SARS-CoV-2 RBD protein and could be tracked along the relevant elution fractions. The full-length spike protein was also expressed (data not shown.)
High-fidelity PTMs for Optimal Functionality
The incorporation of essential PTMs in the SARS-CoV-2 RBD was confirmed using a combination of analytical techniques.
Glycosylation was assessed via SDS-PAGE mobility shifts after glycosidase treatment with Peptide:N-glycosidase F (PNGase F) or Endoglycosidase H (EndoH). Similar migration differences observed with both glycosidases indicated the predominance of high-mannose and hybrid N-glycans.
Further in-depth glycan analysis was performed using liquid chromatography- electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS.) This analysis revealed a high degree of N-glycosylation occupancy across all N-glycosites, with a dominant mannose 8 (Man8) species. Plant-specific glycosylation patterns, such as α1,3-fucosylation and β1,2-xylosylation, were also detected, particularly within the RBD protein.
Disulfide bond formation was confirmed via pepsin digestion, verifying all four pairs of disulfide bonds.
RBD Binding Assays show Recognition by Antibodies and Patient Serum
Binding assays confirmed the ability of the ALiCE®-produced SARS-CoV-2 RBD to maintain its biological function. Using ELISA plates coated with RBD proteins, antibodies targeting conformational (MAB10540) and linear (MAB105802) epitopes demonstrated equivalent binding signals for RBDs produced in both ALiCE® and HEK293 systems. Reactivity against the full S1 protein was observed to be lower, underscoring the specificity of the RBD-focused response.
The biological relevance of ALiCE®-produced RBD was further validated using patient serum samples. Serum from COVID-19 patients with positive PCR tests collected in 2020 showed clear reactivity to the RBD antigen, while serum from pre-2019 patients served as negative controls. A HaloTag-GST fusion protein was used as an additional negative control. The results demonstrated that the serological reactivity of ALiCE®-produced RBD was comparable to that of HEK293-produced RBD.
KEY BENEFITS OF ALiCE®
The rapid expression timelines with ALiCE® enable the production of antigens within 48 hours. This enhanced speed allows rapid testing of multiple constructs to identify variants with the strongest immune response or antibody binding activity. ALiCE®’s flexibility makes it ideal for evolving needs, such as evaluating antibodies against emerging viral strains for diagnostic and therapeutic purposes.
Consistent PTMs and Functionality
The SARS-CoV-2 RBD expressed in ALiCE® maintains a consistent glycan profile, comparable to HEK-derived RBD, with correct disulfide bond formation and high purity. These qualities make ALiCE®-produced antigens suitable for vaccine and diagnostic applications without compromising on functionality.
Pandemic Preparedness and Distributed Medicine Manufacturing
As a eukaryotic cell-free system, ALiCE® can express proteins that are challenging for traditional systems, including toxic antigens or those requiring complex PTMs. This capability ensures the reliable production of high-quality antigens suitable for vaccine development.
The platform’s scalability and efficiency also present opportunities to support pandemic preparedness and for individualized vaccines and distributed medicine manufacturing. This could transform the way we respond to future viral threats.
Moreover, the quality of the VLPs produced using ALiCE®, as shown by TEM analysis and immune response assays, demonstrates its effectiveness as a VLP factory. This makes ALiCE® a versatile and reliable platform for vaccine production in response to evolving threats
Future Outlook
The ALiCE® system is now being actively explored for scaling production of SARS-CoV-2 antigens for pre-clinical trials as part of a CEPI-funded project. These initial findings highlight its ability to generate high-quality antigens with consistent PTMs, making it suitable for vaccine development and large-scale applications.
This versatility underscores ALiCE®’s potential to address the “100-days mission,” a global initiative aimed at accelerating vaccine development timelines to mitigate the impact of future pandemics. By enabling rapid testing and production of multiple antigen variants, ALiCE® can streamline pandemic preparedness and therapeutic development.
We have achieved several important milestones, including the validation of Touchlight’s enzymatic dbDNA™ technology for ALiCE® reactions, which ensures streamlined initiation of protein expression processes.
References
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