Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System

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 pastorisbased 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.

Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation

“…It is worth to note that the achieved production yields of both HCV core‐HBc VLP and EBNA1‐HBc VLP are significantly higher than other reported HBc VLPs of 6.4 mg/L produced using cell‐free protein synthesis system [51] and 3.21 mg/L produced using E. coli expression system in IBs [52]. In addition, the achieved production yields are also higher than the reportedly high production yield in Pichia pastoris of 3 mg/g of wet cell weight [30].…”

In Vitro Assembly of Virus-Like Particles and Their Applications

“…Lastly, cell-free protein synthesis systems have also been used to produce viral capsid proteins in defined transcription/translation reactions. This method allows for a direct control of capsid protein expression and VLP assembly conditions and enables production of toxic and insoluble proteins [25][26][27][28].…”

A Comprehensive Overview on the Production of Vaccines in Plant-Based Expression Systems and the Scope of Plant Biotechnology to Combat against SARS-CoV-2 Virus Pandemics

“…Many reports explain the role of NPs in SARS-CoV-2 in detail [136][137][138][139][140][141][142]. For the production of VLPs, several studies target Poliovirus, hepatitis B virus, human papillomavirus, influenza virus, Norwalk virus, human immunodeficiency retrovirus 1, rift valley fever virus, and foot and mouth disease virus [132,[146][147][148][149][150][151][152][153][154][155][156][157][158]. Earlier experience of forming VLPs for MERS and SARS-CoV-1 antigens heterologous expressed in recombinant systems provides us the best platform for developing a vaccine against SARS-CoV-2.…”