1. Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis.
- Author
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Deuker D, Asilonu E, Bracewell DG, and Frank S
- Subjects
- Humans, HeLa Cells, Capsid Proteins genetics, Capsid Proteins metabolism, Capsid Proteins biosynthesis, Protein Biosynthesis, Virion genetics, Virion metabolism, Escherichia coli genetics, Escherichia coli metabolism, Dependovirus genetics, Cell-Free System
- Abstract
Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli -based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of ∼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli -based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis.
- Published
- 2024
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