Abstract
BackgroundThe last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags.ResultsWe designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein.ConclusionFour newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.
Highlights
The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics
Various sources of translation machinery can be used for cellfree in vitro translation systems, but -due to its low cost and capacity for synthesizing properly folded, high molecular weight eukaryotic proteins- wheat germ derived protein extract presently seems the most promising choice [4]
We demonstrated that a protein kinase purified from a wheat germ in vitro translation reaction possessed higher in vitro kinase activity than the same kinase produced by overexpression in E. coli
Summary
The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Accelerated proteomics studies demand high-throughput protein production methods to ensure availability of proteins of interest. Overexpression in E. coli cells is the most preferred protein production method Though this system has been well optimized and is suitable for the simultaneous generation of a panel of proteins, its application is often limited by the insolubility of synthesized eukaryotic proteins [1]. Various sources of translation machinery can be used for cellfree in vitro translation systems, but -due to its low cost and capacity for synthesizing properly folded, high molecular weight eukaryotic proteins- wheat germ derived protein extract presently seems the most promising choice [4]
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