Abstract
Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.
Highlights
Cell-free protein synthesis was developed as early as the 1960s, where it was critical in determining the genetic code (Nirenberg and Matthaei, 1961)
In this study we have developed a rapid method for the production and partial purification of Hepatitis B virus core antigen (HBc) virus-like particle (VLP) produced in a P. pastoris cell-free system
The full-length wild-type HBc VLP was selected as a proof-ofconcept due to extensive previous characterization and its use as a chassis molecule for novel vaccine designs (Pumpens and Grens, 2001)
Summary
Cell-free protein synthesis was developed as early as the 1960s, where it was critical in determining the genetic code (Nirenberg and Matthaei, 1961). Advances in synthetic biology are driving a renaissance in CFPS and it is emerging as a transformative platform technology (Carlson et al, 2012). This pique in interest is mainly due to the capacity to produce protein in a rapid and facile manner, providing benefits to multifarious applications. These include the design of de novo metabolic pathways (Hodgman and Jewett, 2012), personalized medicine (Ogonah et al, 2017) and biosensing (Pardee et al, 2016). Due Abbreviations: CFPS, cell-free protein synthesis; CIP, calf-intestinal alkaline phosphatase; CTD, C-terminal domain; DLS, dynamic light scattering; HBc, hepatitis B core antigen; MWCO, molecular weight cut-off; NTD, N-terminal domain; OD600, optical density at 600 nm; TEM, transmission electron microscopy; VLP, virus-like particle
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