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.
The authors describe siblings with progressive external ophthalmoplegia (PEO) due to a novel heterozygous A to G transition at nucleotide 955 of C10Orf2 (Twinkle). The mutation was not identified in parents' blood, hair follicles, buccal mucosa, or urinary epithelium, indicating germ line mosaicism. One sibling presented with sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO), a phenotype previously associated with the POLG1 gene, highlighting the clinical overlap in autosomal PEO.
While the deletion of an amino acid is a common mutation observed in nature, it is generally thought to be disruptive to protein structure. Using a directed evolution approach, we find that the enzyme TEM-1 b-lactamase was broadly tolerant to the deletion mutations sampled. Circa 73% of the variants analysed retained activity towards ampicillin, with deletion mutations observed in helices and strands as well as regions important for structure and function. Several deletion variants had enhanced activity towards ceftazidime compared to the wild-type TEM-1 demonstrating that removal of an amino acid can have a beneficial outcome.
Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeqNNN. The bla gene encoding TEM-1 β-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.
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