ABSTRACT
High-yield production of soluble protein is a common concern in diverse fields of biotechnology. In this study, a strategy of using an engineered nucleotide sequence of ubiquitin for enhancing the production of soluble proteins in a cell-free synthesis system is presented. When examined for a series of proteins that otherwise were poorly expressed, N-terminal fusion with ubiquitin significantly increased both the expression levels and solubility of the translational products. The effect of ubiquitin fusion was also markedly augmented by engineering the nucleotide sequence of ubiquitin, leading to several fold enhancements in soluble production of target proteins. Recombinant proteins were produced with their native amino acid sequences through in situ removal of ubiquitin during cell-free synthesis reactions in the presence of a deubiquitinase. The presented strategy could be employed as a facile route to prepare soluble proteins required for various applications.
Subject(s)
Cell-Free System/metabolism , Protein Engineering/methods , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Folding , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Solubility , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs. It is generated nonribosomally from a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized. Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.
