Tuning the Cell-Free Protein Synthesis System for Biomanufacturing of Monomeric Human Filaggrin

Jeehye Kim,Bastian Vögeli,Kosuke Seki,Yong-Chan Kwon,Caroline E. Copeland

doi:10.3389/fbioe.2020.590341

Abstract

The modern cell-free protein synthesis (CFPS) system is expanding the opportunity of cell-free biomanufacturing as a versatile platform for synthesizing various therapeutic proteins. However, synthesizing human protein in the bacterial CFPS system remains challenging due to the low expression level, protein misfolding, inactivity, and more. These challenges limit the use of a bacterial CFPS system for human therapeutic protein synthesis. In this study, we demonstrated the improved performance of a customized CFPS platform for human therapeutic protein production by investigating the factors that limit cell-free transcription–translation. The improvement of the CFPS platform has been made in three ways. First, the cell extract was prepared from the rare tRNA expressed host strain, and CFPS was performed with a codon-optimized gene for Escherichia coli codon usage bias. The soluble protein yield was 15.2 times greater with the rare tRNA overexpressing host strain as cell extract and codon-optimized gene in the CFPS system. Next, we identify and prioritize the critical biomanufacturing factors for highly active crude cell lysate for human protein synthesis. Lastly, we engineer the CFPS reaction conditions to enhance protein yield. In this model, the therapeutic protein filaggrin expression was significantly improved by up to 23-fold, presenting 28 ± 5 μM of soluble protein yield. The customized CFPS system for filaggrin biomanufacturing described here demonstrates the potential of the CFPS system to be adapted for studying therapeutic proteins.

Highlights

Owing to the open nature of the cell-free biology, the cell-free protein synthesis (CFPS) system provides practicable opportunities to design and re-design the cellular processes outside the cell (Silverman et al, 2019)
Recent advances in the bacterial CFPS systems have overcome this technological barrier and equipped the synthetic capability of the protein therapeutics. i) The modern CFPS system has opened the opportunity of cell-free biomanufacturing of vaccines and personalized protein therapeutics by enabling cell-free protein glycosylation (Guarino and DeLisa, 2012; Jaroentomeechai et al, 2018; Kightlinger et al, 2018, 2020). ii) The synthesis of membrane proteins, which typically are a target of many drugs in clinical studies, has been improved by the addition of membrane mimics such as peptide surfactants (Wang et al, 2011), membrane fragments (Shinoda et al, 2016), and liposomes (Matthies et al, 2011). iii) The on-demand vaccine productions have been achieved in the CFPS system as well
The cytosol-penetrating antibodies have been synthesized in the CFPS system (Min et al, 2016). v) There have been improvements in the CFPS systems devoted to the cancer therapeutic proteins such as onconase (Salehi et al, 2016) and crisantaspase (Hunt et al, 2019), demonstrating the CFPS platform to be advanced and on-demand technology for future cancer therapeutics

Summary

Introduction

Owing to the open nature of the cell-free biology, the cell-free protein synthesis (CFPS) system provides practicable opportunities to design and re-design the cellular processes outside the cell (Silverman et al, 2019). Cell-Free Filaggrin Biomanufacturing translation can be precisely tuned in an open environment of the in vitro platform, which is hard to achieve in the living system (Lu, 2017). The CFPS system takes its strong position in the race for the next-generation biomanufacturing platform as well as opens a new avenue for the precise synthesis of protein therapeutics. Recent advances in the bacterial CFPS systems have overcome this technological barrier and equipped the synthetic capability of the protein therapeutics. I) The modern CFPS system has opened the opportunity of cell-free biomanufacturing of vaccines and personalized protein therapeutics by enabling cell-free protein glycosylation (Guarino and DeLisa, 2012; Jaroentomeechai et al, 2018; Kightlinger et al, 2018, 2020). The cytosol-penetrating antibodies have been synthesized in the CFPS system (Min et al, 2016). v) There have been improvements in the CFPS systems devoted to the cancer therapeutic proteins such as onconase (Salehi et al, 2016) and crisantaspase (Hunt et al, 2019), demonstrating the CFPS platform to be advanced and on-demand technology for future cancer therapeutics

Methods

Results

Conclusion