Keywords

cell-free protein synthesis, virus-like particle, Qβ VLP, CalB, Candida antarctica lipase B, enzyme stabilization

Abstract

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step.

Original Publication Citation

Yang, S. O., Talley, J. P., Nielsen, G. H., Wilding, K. M., & Bundy, B. C. (2025). Streamlined Production, Protection, and Purification of Enzyme Biocatalysts Using Virus-like Particles and a Cell-Free Protein Synthesis System. SynBio, 3(1), 5. https://doi.org/10.3390/synbio3010005

Document Type

Peer-Reviewed Article

Publication Date

2025-02-05

Publisher

SynBio

Language

English

College

Ira A. Fulton College of Engineering

Department

Chemical Engineering

University Standing at Time of Publication

Full Professor

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