Construction of a random circular permutation library using an engineered transposon

Brennal Pierre, Vandan Shah, Jenny Xiao, Jin Kim

Research output: Contribution to journalArticle

Abstract

Circular permutation is an important protein engineering tool used to create sequence diversity of a protein by changing its linear order of amino acid sequence. Circular permutation has proven to be effective in the evolution of proteins for desired properties while maintaining similar three-dimensional structures. Due to the lack of a robust design principle guiding the selection of new termini, construction of a combinatorial library is much preferred for comprehensive evaluation of circular permutation. Unfortunately, the conventional methods used to create random circular permutation libraries cause significant sequence modification at new termini of circular permutants. In addition, these methods impose additional limitations by requiring either relatively inefficient blunt-end ligation during library construction or redesign of transposons for tailored expression of circular permutants. In this study, we present the development of an engineered transposon for facile construction of random circular permutation libraries. We provide evidence that minimal modification at the new termini of the random circular permutants is possible with our engineered transposon. In addition, our method enables the use of sticky-end ligation during library construction and provides external tunability for expression of random circular permutants.

Original languageEnglish (US)
Pages (from-to)16-24
Number of pages9
JournalAnalytical Biochemistry
Volume474
DOIs
StatePublished - Apr 1 2015

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Libraries
Ligation
Protein Engineering
Proteins
Amino Acid Sequence
Amino Acids

Keywords

  • Circular permutation
  • Combinatorial library
  • Protein engineering
  • Transposon

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Construction of a random circular permutation library using an engineered transposon. / Pierre, Brennal; Shah, Vandan; Xiao, Jenny; Kim, Jin.

In: Analytical Biochemistry, Vol. 474, 01.04.2015, p. 16-24.

Research output: Contribution to journalArticle

Pierre, Brennal ; Shah, Vandan ; Xiao, Jenny ; Kim, Jin. / Construction of a random circular permutation library using an engineered transposon. In: Analytical Biochemistry. 2015 ; Vol. 474. pp. 16-24.
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