Design of a Split Intein with Exceptional Protein Splicing Activity

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• 作者：Adam J. Stevens ; Zachary Z. Brown ; Neel H. Shah ; Giridhar Sekar ; David Cowburn ; Tom W. Muir
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：February 24, 2016
• 年：2016
• 卷：138
• 期：7
• 页码：2162-2165
• 全文大小：289K
• ISSN：1520-5126

文摘

Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques.