Abstract
One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.
Highlights
Systems biology seeks to understand genomic and proteomic changes between species or between individual cells to link compositional changes in the genome to the observed phenotype
All cell-free translation systems rely on two major components: (1) a designed vector or a polymerase chain reaction (PCR) template with your gene of interest, and (2) a cell extract of Novikova et al Adv Struct Chem Imag (2018) 4:13 choice that matches the experimental needs for yield or post-translational modifications
Quick translation trials to determine the protein expression potential (MINI) PCR templates for cell-free expression can be prepared in short amount of time and can, be used to quickly test target protein for expression and solubility
Summary
Systems biology seeks to understand genomic and proteomic changes between species or between individual cells to link compositional changes in the genome (or proteome) to the observed phenotype. The reagents are supplemented with additional energy sources, amino acids and various cofactor molecules to aid continuous protein synthesis and folding Key advantages of these systems are the ease of the setup, fast turnaround times, linear scalability for the reaction volumes and amenability to high-throughput screening. Since there is no need to sustain a living organism, toxic proteins that failed to be produced by cell-based methods can be readily expressed [23] These numerous benefits are being widely exploited in applications of NMR and X-ray structure determination, functional genomics and proteome research, protein chip construction, therapeutics and vaccine development [17, 23,24,25,26]
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