A general method for scanning unnatural amino acid mutagenesis

Abstract

Current approaches to protein site-directed mutagenesis require an independent user operation for each mutation. This can impede large-scale scanning mutagenesis projects such as the mapping of protein interaction surfaces, active sites, or epitopes. It also prevents the creation of protein libraries of defined complexity for directed evolution purposes. Here we present a simple, fast, and effective way to perform scanning codon mutagenesis throughout a protein sequence. The process allows the researcher to define the new codon change, and therefore any amino acid mutation can be achieved. We demonstrate this approach by creating a library of proteins that contain single unnatural amino acid mutations encoded by the amber stop codon, TAG. The mutant proteins generated by this method can be expressed and assayed individually or used together as a mixed population of "rationally diversified" protein sequences.

Figure 1

Schematic diagram of Codon Scanning Mutagensis. a. A plasmid containing the gene (blue) to be mutated is first targeted with a modified Mu transposon (green) that is inserted in vitro by the action of MuA transposase, resulting in a library of plasmids that are recovered in E. coli. This insertion event results in the duplication of five nucleotides, N₁N₂N₃N₄N₅, at the site of insertion (shown in b). The library can be purified to only contain members where the transposon was inserted into the gene of interest by restriction digest. b. The transposon fragment with recognition sequences, R1 and R2, is modified to have MlyI restirciton sites on either end. Three base pairs, N₂N₃N₄ of the original plasmid sequence is then removed by digestion with MlyI, and (c.) a new frame selectable TAG linker segment of DNA (orange) is ligated into this digestion site, resulting in a library of plasmids that are selected for correct reading frame in E. coli. The basis for the reading frame selection is assembly of a functional β-lactamase-intein chimera which splices to yield colonies resistant to ampicillin. This library is then subjected to a second digestion and re-ligated, thus resulting in (d.) selective removal of the linker and a net replacement of a new codon.

Figure 2

Sequence of a single clone proceeding through the process. a. Sequence of wild-type glutathione S-transferase, the highlighted ACA is the codon removed by MlyI digestion of the transposon. b. The linker is inserted in-frame resulting in ampicillin resistance, the MlyI restriction sites are highlighted and red arrows indicate the cut sites. Upon digestion with MlyI and subsequent ligation the codon TAG (shown in yellow c.) replaces ACA.

Figure 3

a. SDS/PAGE analysis of expression of 10 amber codon mutants in the presence (+) and absence (-) of pBpa. Amino acid residues mutated to TAG codons are shown. One mutant, L65TAG, did not express for unknown reasons. b. Photo-activity of high expression mutants derived from scanning compared to a mutant known to cross-link (F51).