Knock-In-Leave-Behind (KILB): Genetic Grafting of Protease-Cleaving Sequences into Permissive Sites of Proteins with a Tn5-Based Transposition System

Abstract

Endowing proteins with proteolytic cleavage sites without affecting their native function when the cognate protease is not present is a challenging engineering effort for fundamental studies and biotechnological applications. Insertion of such short polypeptides often requires some knowledge of the target protein structure or identification of permissive sites that accept the genetic grafting without loss of function, e.g., by means of transposon-mediated linker-scanning mutagenesis. We describe a procedure to deliver in-frame polypeptides throughout the sequence of any target protein with a knock-in-leave-behind (KILB) transposon-based method. The mini-Tn5 synthetic transposable element reported here was tailored to randomly introduce recognition sites of the specific viral protease NIa into permissive locations of the target protein. Protein insertion variants can then be examined to detect phenotypic differences once cleaved in vivo by the cognate protease. Two application scenarios are discussed, i.e., proteolizable variants of the regulatory protein XylR of Pseudomonas putida and development of phenotypic mutants of metabolic functions.