Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.
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 beta-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b(562) 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 alpha-helices and beta-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.
School of Biosciences and School of Chemistry, Cardiff University, Cardiff, UK.
Wayne R
WR
Edwards
Kathy
K
Busse
Rudolf K
RK
Allemann
D Dafydd
DD
Jones
D003573:Cytochrome b Group / Q000235:genetics; D004251:DNA Transposable Elements; D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D006418:Heme / Q000378:metabolism; D008958:Models, Molecular; D019897:Periplasm / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry*; D017384:Sequence Deletion; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003573:Cytochrome b Group; D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; D006418:Heme; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1