(157av) Synthesis and Application of Cell-Free Synthesized Therapeutic Human Proteins

Proteins are highly structural polymers, performing an enormous range of roles within organisms. Unlike synthetic polymers, protein has a significant hierarchical ordering and complexity, which hinders it from being produced and used in a therapeutic purpose. Recent advancement in synthetic biology has been applied to high yielding protein production where cell extract from clarified cell lysate is used as transcription and translational machinery for protein synthesis ex vivo. Cell-Free Protein Synthesis (CFPS) is an advanced biosynthetic method for protein production with the ease of system manipulation for protein engineering. It is also more flexible in protein manufacturing even when the conventional cell-based protein synthesis is not efficient (e.g. antibiotics and anticancer peptides that are unfavorable to the growth of host organisms).

In this study, human skin protein Filaggrin (FLG) was selected as a model therapeutic protein. Even though its role in restoring the skin barrier integrity in the skin of patients is significant, the use of FLG as a treatment has not been studied much yet. This study aimed to increase production yield (protein mass per volume) in prokaryotic CFPS. First, the yield has increased in CFPS by optimizing (1) bacteria codon usage (codon optimization, use of strain encoding rare codon tRNA), and (2) protein synthesis condition (temperature, reaction buffer). Next, the soluble protein and total protein were quantified in both optimized CFPS and cell-based protein synthesis. The CFPS showed great potential to produce therapeutic proteins.