Ribosomal Protein S1 Improves the Protein Yield of an In Vitro Reconstituted Cell-Free Translation System

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biochemistry

Background:

• Cell-free transcription-translation (TX-TL) systems enable biological engineering outside living cells.
• Current TX-TL systems face limitations in ribosome function, leading to low protein yields.
• Ribosomal protein S1's variable presence affects translation initiation and mRNA recruitment.

Purpose of the Study:

• To investigate the impact of supplementing a reconstituted TX-TL system with ribosomal protein S1.
• To determine if S1 supplementation can enhance protein yields in cell-free expression.
• To explore the effect of S1 on translation initiation and ribosome functionality.

Main Methods:

• Utilized a commercial in vitro reconstituted TX-TL system (PURExpress).
• Supplemented the system with purified recombinant ribosomal protein S1.
• Assessed protein yields using various reporter proteins (EYFP, sfGFP, mRFP) and 5'UTR contexts.
• Evaluated expression of a structured RNA (PSIV IRES).

Main Results:

• Supplementation with purified recombinant S1 doubled protein yields in the TX-TL system.
• Improved yields were observed across different reporter proteins and 5'UTR sequences.
• Enhanced expression was achieved even for highly structured RNAs like PSIV IRES.
• Fine-tuning S1 concentration optimized protein production.

Conclusions:

• Ribosomal protein S1 is a critical factor for enhancing TX-TL system performance.
• Supplementing S1 increases the fraction of functional ribosomes, boosting translation efficiency.
• Optimizing S1 concentration offers a novel strategy to improve cell-free protein synthesis yields.