Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

Abstract

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.

Transposon for protein engineering

“…22,23 Transposons for random insertional fusion of protein domains Insertional protein fusion is an advanced engineering approach to generate novel proteins with integrated functions. Three critical parameters that can determine the success of functional integration between fused proteins are the insertion location within the host protein, 24,25 the inter-domain linker length 26,27 and the inter-domain linker composition. 26,28 Interdomain linker length and composition should be carefully designed to avoid a steric hindrance between the 2 fused domains and to facilitate maximum interdomain interactions.…”

“…Three critical parameters that can determine the success of functional integration between fused proteins are the insertion location within the host protein, 24,25 the inter-domain linker length 26,27 and the inter-domain linker composition. 26,28 Interdomain linker length and composition should be carefully designed to avoid a steric hindrance between the 2 fused domains and to facilitate maximum interdomain interactions. 29 Among various methods for generating protein insertional fusions, a combinatorial approach is especially beneficial because robust guidelines for selection of insertion sites are unavailable.…”

“…[34][35][36] In another method, control of inter-domain linker length and composition is made possible by the removal of an inserted transposon from a host DNA sequence after transposition. 26 However, similar to the endonuclease-based methods, this transposonbased method depends on the blunt-end ligation between host and guest DNAs.…”

An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions

“…24,34,35 This minimal in vitro reaction has proven to be highly efficient, and it has a low target site selectivity. 24,36,37 To date, Mu transposition reaction has been utilized in a variety of molecular biology, [38][39][40][41][42][43][44][45][46][47] protein engineering, [48][49][50][51][52] and genomics applications, 10,[53][54][55] and also for efficient gene delivery in bacteria, yeast, and mammalian cells. [56][57][58][59] Although in vitro transposition reactions have been widely used in various applications, a universal activity measurement assay to quantify transpositional activity is lacking.…”

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons

“…Notably, Jones and co-workers described a Mu transposon variant in which the flanking sequences were modified to contain the recognition sites of MlyI, a type IIS restriction endonuclease (Jones, 2005). Upon MlyI digestion, the transposon sequence can be removed from the target gene along with a triplet nucleotide that can be subsequently replaced by three new nucleotides (Baldwin et al, 2008) or even a new protein domain (Edwards et al, 2008). This approach, referred to as triplet nucleotide exchange (TriNEx), is perhaps the closest to achieve a non-redundant mutagenesis method (Baldwin et al, 2008).…”