title: Facile construction of a random protein domain insertion library using an engineered transposon.
abstract: Insertional fusion between multiple protein domains represents a novel means of creating integrated functionalities. Currently, there is no robust guideline for selection of insertion sites ensuring the desired functional outcome of insertional fusion. Therefore, construction and testing of random domain insertion libraries, in which a host protein domain is randomly inserted into a guest protein domain, significantly benefit extensive exploration of sequence spaces for insertion sites. Short peptide residues are usually introduced between protein domains to alleviate structural conflicts, and the interdomain linker residues may affect the functional outcome of protein insertion complexes. Unfortunately, optimal control of interdomain linker residues is not always available in conventional methods used to construct random domain insertion libraries. Moreover, most conventional methods employ blunt-end rather than sticky-end ligation between host and guest DNA fragments, thus lowering library construction efficiency. Here, we report the facile construction of random domain insertion libraries using an engineered transposon. We show that random domain insertion with optimal control of interdomain linker residues was possible with our engineered transposon-based method. In addition, our method employs sticky-end rather than blunt-end ligation between host and guest DNA fragments, thus allowing for facile construction of relatively large sized libraries.
authors.affiliation: Othmer-Jacobs Department of Chemical and Biomolecular Engineering, Polytechnic Institute of New York University, Brooklyn, NY 11201, USA.
authors.forename: Vandan
authors.initials: V
authors.lastname: Shah
authors.forename: Brennal
authors.initials: B
authors.lastname: Pierre
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D015698:Genomic Library; D016254:Mutagenesis, Insertional / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary / Q000235:genetics; D019895:Transposases / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases