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Abstract
The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.
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Details
1 Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore
2 Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
3 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore; Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
4 Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Translational Laboratory in Genetic Medicine, Singapore, Singapore; Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore