title: Construction of a random circular permutation library using an engineered transposon.
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.
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, Brooklyn, NY 11201, USA.
authors.forename: Brennal
authors.initials: B
authors.lastname: Pierre
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, Brooklyn, NY 11201, USA.
authors.forename: Vandan
authors.initials: V
authors.lastname: Shah
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, Brooklyn, NY 11201, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, New York University, Brooklyn, NY 11201, USA. Electronic address: jin.kim@nyu.edu.
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D010957:Plasmids / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004270:DNA, Circular
keywords: Circular permutation; Combinatorial library; Protein engineering; Transposon