Design of a Split Intein with Exceptional Protein Splicing Activity

Adam J. Stevens, Zachary Z. Brown, Neel H. Shah, Giridhar Sekar, David Cowburn, Tom W. Muir

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2162-2165
Number of pages4
JournalJournal of the American Chemical Society
Volume138
Issue number7
DOIs
StatePublished - Mar 2 2016

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Inteins
Protein Splicing
Proteins
Trans-Splicing
Mutagenesis
Sequence Alignment
Consensus Sequence
Biotechnology
Antibodies
Catalytic Domain
Durability
Fusion reactions
Hot Temperature
Technology
Engineers

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Design of a Split Intein with Exceptional Protein Splicing Activity. / Stevens, Adam J.; Brown, Zachary Z.; Shah, Neel H.; Sekar, Giridhar; Cowburn, David; Muir, Tom W.

In: Journal of the American Chemical Society, Vol. 138, No. 7, 02.03.2016, p. 2162-2165.

Research output: Contribution to journalArticle

Stevens, Adam J. ; Brown, Zachary Z. ; Shah, Neel H. ; Sekar, Giridhar ; Cowburn, David ; Muir, Tom W. / Design of a Split Intein with Exceptional Protein Splicing Activity. In: Journal of the American Chemical Society. 2016 ; Vol. 138, No. 7. pp. 2162-2165.
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