From Electronic Sequence to Purified Protein Using Automated Gene Synthesis and In Vitro Transcription/Translation

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• De novo gene synthesis is crucial for synthetic biology but downstream protein production remains challenging.
• Traditional recombinant protein production involves extensive, time-consuming steps like cloning and cell culture.
• Limited progress exists in automating protein expression from synthetic genetic material.

Purpose of the Study:

• To develop a fast, automated workflow for cell-free protein production from electronic protein sequences.
• To streamline the process from gene sequence to functional protein, reducing hands-on time and accelerating research.
• To enable high-throughput production of synthetic proteins for various applications.

Main Methods:

• Developed PRESTO (protein expression starting from oligonucleotides), an automated workflow.
• Integrated in silico sequence optimization, oligonucleotide assembly into DNA expression cassettes, and in vitro transcription/translation.
• Utilized a small liquid handling system for a hands-free, high-throughput process.

Main Results:

• Achieved cell-free protein production starting from electronic protein sequences or accession numbers.
• Successfully combined sequence optimization, DNA assembly, in vitro expression, and purification in one automated workflow.
• Delivered functional synthetic proteins within 1 day, demonstrating a significant acceleration of the protein production pipeline.

Conclusions:

• PRESTO offers a rapid and automated solution for cell-free protein production, overcoming limitations of traditional methods.
• This workflow significantly accelerates the generation of functional synthetic proteins, supporting advancements in synthetic biology and biotechnology.
• The hands-free, high-throughput nature of PRESTO makes it a valuable tool for researchers needing rapid access to custom proteins.