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

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Cell-free protein synthesis (CFPS) offers a versatile platform for biomanufacturing therapeutic proteins.
• Bacterial CFPS systems face challenges in producing active human proteins, including low expression, misfolding, and inactivity.
• Overcoming these limitations is crucial for advancing cell-free biomanufacturing of human therapeutics.

Purpose of the Study:

• To improve the performance of a customized CFPS platform for enhanced human therapeutic protein production.
• To investigate and address the limiting factors in bacterial cell-free transcription-translation for human protein synthesis.
• To optimize CFPS conditions for increased yield and activity of therapeutic proteins.

Main Methods:

• Prepared cell extract from a host strain overexpressing rare tRNAs.
• Utilized codon-optimized genes for Escherichia coli codon usage bias in the CFPS system.
• Identified critical biomanufacturing factors and engineered reaction conditions to enhance protein yield.

Main Results:

• Achieved a 15.2-fold increase in soluble protein yield using rare tRNA overexpressing host extract and codon-optimized genes.
• Significantly improved the expression of the therapeutic protein filaggrin by up to 23-fold.
• Obtained a soluble protein yield of 28 ± 5 μM for filaggrin.

Conclusions:

• The customized CFPS platform demonstrates significantly improved performance for human therapeutic protein production.
• Optimizing cell extract composition and gene codon usage enhances soluble protein yield in bacterial CFPS.
• This engineered CFPS system shows potential for the efficient biomanufacturing and study of therapeutic proteins.