Abstract
We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.
Highlights
Finding the optimal host for the expression of proteins for research purposes often involves expensive and time-consuming testing in multiple expression systems
We have developed a new set of expression vectors, named the pONE series, suitable for protein expression in various, common host organisms
A unified and modular vector set for recombinant protein expression expression cassette, which allows rapid expression screening of novel proteins, and easy manipulation of fusion tags and signal peptides
Summary
Finding the optimal host for the expression of proteins for research purposes often involves expensive and time-consuming testing in multiple expression systems. Since different vectors were developed independently [1,2,3], they are usually not compatible, holding a different set of restriction sites. This means that in many cases the gene of interest needs to be re-cloned into each vector to be tried, even if the gene itself is unchanged. The expression screening process can be sped up by the use of vectors tailored/designed for quick exchange of genes of interest. One such approach is the pAUL vector system developed for Arabidopsis thaliana [4]. Other systems are aimed to handle different problems
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