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

• DOI: 10.1021/acssynbio.0c00060
• Publication Date: 2020-06-05T21:45:16Z
• Authors: Claudia Chiocchini; Krishna Vattem; Michael Liss; Lisa Ludewig; Tobias Reusch; Ischwaku Rastogi; Brian Webb; Axel Trefzer
• Keywords: 0301 basic medicine; Base Sequence; Transcription, Genetic; Green Fluorescent Proteins; Oligonucleotides; CHO Cells; Recombinant Proteins; 03 medical and health sciences; Cricetulus; Protein Biosynthesis; Escherichia coli; Animals; Humans; Computer Simulation; Streptokinase; Synthetic Biology; Amino Acid Sequence; Luciferases; Erythropoietin

De novo gene synthesis is the state-of-the-art method used to obtain genetic material adapted to the requirements of the host organism and a cornerstone for modern synthetic biology. Yet, little progress has been made regarding downstream processes of protein production from synthetic genetic material. The production of recombinant proteins traditionally requires extensive preparatory work including gene amplification, cloning, sequencing, transformation or transfection of the expression host, cultivation of living cells, and purification of the overexpressed protein. In this work we describe a fast and automated workflow for cell-free production of proteins starting from an electronic protein sequence or accession number. PRESTO (protein expression starting from oligonucleotides) seamlessly combines a tailored in silico sequence optimization with the assembly of short oligonucleotides into synthetic linear DNA expression cassettes, mammalian in vitro transcription/translation, and protein purification thereof. Integrated on a small liquid handling system it provides a hands-free high throughput source for functional synthetic proteins within 1 day.