Engineered transposon for facile construction of a random protein domain insertion library

摘要

Methods for facile construction of a random domain insertion library (1) with optimal control of composition and length of inter-domain linker residues and (2) mediated by sticky-end ligation between host and guest DNA fragments. To develop such a method, we engineered a Mu transposon. The method exploits transposition of the engineered Mu transposon, which, upon removal, allows for sticky-end ligation between host and guest DNA fragments. We used a gene coding for xylanase from bacillus circulans (BCX) as a guest DNA sequence and the plasmid PUC19 containing lacZα as the target for insertion (i.e., a host DNA sequence). Results demonstrate that the method enables facile construction of a random domain insertion library with optimal control of composition and length of inter-domain linker residues.

主权项

1. A method for facile construction of a random domain insertion library comprising the steps of:
providing an engineered transposon; randomly transposing the engineered transposon into a host DNA sequence, using plasmid PUC19 containing lacZα as the host DNA sequence; employing sticky-end ligation between the host DNA sequence and a guest DNA sequence, using a gene coding for xylanase from bacillus circulans (BCX) as the guest DNA sequence; and removing the engineered transposon from the host DNA sequence by restriction enzyme digestion, wherein the restriction enzyme digestion is double digestion using BclI and AgeI restriction enzymes, thereby generating a plasmid for the domain insertion library.