期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 86, 期 2, 页码 83-88出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.09.012
关键词
Protein library design; High-quality libraries; Synthetic biology; Post-translational modification
类别
资金
- Howard Hughes Medical Institute program (GCPALS)
- University of Kentucky Office of Undergraduate Research
- NIH National Center for Research Resources (NCRR) [P20 RR020171]
- Kentucky Science and Engineering Foundation [KSEF-148-502-207-201]
- Kentucky Science and Technology Corporation
- University of Kentucky College of Medicine Startup funds
By successfully incorporating sequence diversity into proteins, combinatorial libraries have been a staple technology used in protein engineering, directed evolution, and synthetic biology for generating proteins with novel specificities and activities. However, these approaches mostly overlook the incorporations of post-translational modifications, which nature extensively uses for modulating protein activities in vivo. As an initial step of incorporating post-translational modifications into combinatorial libraries, we present a bacterial co-expression system, utilizing a recently characterized calmodulin methyltransferase (CaM KMT), to trimethylate a combinatorial library of the calmodulin central linker region. We show that this system is robust, with the successful over-expression and post-translational modification performed in Escherichia coli. Furthermore we show that trimethylation differentially affected the conformational dynamics of the protein upon the binding of calcium, and the thermal stability of the apoprotein. Collectively, these data support that when applied to an appropriately designed protein library scaffold, CaM KMT is able to produce a post-translationally modified library of protein sequences, thus providing a powerful tool for future protein library designs and constructions. (C) 2012 Elsevier Inc. All rights reserved.
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