Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis
- 30 January 2012
- journal article
- letter
- Published by American Chemical Society (ACS) in ACS Synthetic Biology
- Vol. 1 (2), 43-52
- https://doi.org/10.1021/sb3000029
Abstract
Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multienzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis.Keywords
This publication has 47 references indexed in Scilit:
- Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymeraseBiotechnology & Bioengineering, 2011
- Cell-free synthetic biology: Thinking outside the cellMetabolic Engineering, 2011
- Enzymatic De Novo Pyrimidine Nucleotide SynthesisJournal of the American Chemical Society, 2010
- A Tissue-Specific Atlas of Mouse Protein Phosphorylation and ExpressionCell, 2010
- Update on designing and building minimal cellsCurrent Opinion in Biotechnology, 2010
- Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein ParticlesMolecular & Cellular Proteomics, 2008
- Pathway Engineered Enzymatic de Novo Purine Nucleotide SynthesisACS Chemical Biology, 2008
- An integrated cell‐free metabolic platform for protein production and synthetic biologyMolecular Systems Biology, 2008
- Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometryNature Methods, 2007
- An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein databaseJournal of the American Society for Mass Spectrometry, 1994