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


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 ?M 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.

SUBMITTER: Kim J 

PROVIDER: S-EPMC7658397 | BioStudies | 2020-01-01

REPOSITORIES: biostudies

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