Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 β-lactamase to an amino acid deletion

Abstract

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 β-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10 000 μg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.

Investigating protein structural plasticity by surveying the consequence of an amino acid deletion from TEM‐1 β‐lactamase

“…We recently described a new transposon-based directed evolution method used to sample single amino acid deletions from the polypeptide backbone by triplet nucleotide removal at random positions in a target DNA sequence (Supplementary Fig. 1 and [12]). The pNOM derived bla gene that encodes the clinically important TEM-1 b-lactamase was the target for mutagenesis using the engineered transposon MuDel that contained a chloramphenicol resistance gene for selection purposes.…”

“…Deletion mutations provide nature with an added source of protein sequence and conformational diversity not sampled by substitutions. Therefore, deletions should not be ignored when designing or artificially evolving proteins with novel properties, especially with the advent of new directed evolution methods with the potential of sampling such mutations [12,20,21].…”

“…1 and [12]). The selection of clones with MuDel inserted within the bla gene of pNOM was performed using a positive selection for chloramphenicol (Cam) resistance followed by a screen for ampicillin (Amp) sensitivity, as described previously [12]. Cells that grew on Cam but not Amp LB agar plates were picked and constituted the BLAD 391 library.…”

“…To provide further insight into the tolerance of a protein to deletion mutations, we have used a recently developed directed evolution method [12] to remove single amino acids at random positions in the antibiotic hydrolysing enzyme, TEM-1 b-lactamase [13]. We show that the majority of deletion mutations sampled were tolerated by TEM-1, including those in helices and strands, and that they had varying effects on the activity of the enzyme, including improvement of the in vivo activity towards a normally poor substrate.…”

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

“…Interestingly, the transposon sequences outside of the tranposase recognition sites (R sites) can be modified to carry essentially any sequence. 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).…”