(599ce) Engineering Circularly Permuted Proteins with Varying Peptide Tags Using Transposon Mutagenesis

Libraries of circularly permuted proteins can be synthesized by using the transposase MuA to insert an artificial transposon into the gene sequence encoding the protein being permuted. This PERMutation Using Transposase Engineering (PERMUTE) approach generates circularly permuted variants of natural proteins without causing deletions in the primary sequence. However, this approach adds eighteen extra residues to the N-terminus of permuted variants and two amino acids to the C-terminus. We have developed artificial transposons for use with PERMUTE that minimize the number of residues added to the termini, and we have been using these transposons to examine the effect of peptide additions on the function of permuted adenylate kinases (AK). We have created AK libraries using our different artificial transposons and selected for variants that complement the growth of Escherichia coli with a temperature-sensitive AK. Bacterial selections of each library identified overlapping but distinct sets of permuted AK genes that function in Escherichia coli, a majority of which had distinct architectures from permuted AK previously discovered in a library encoding variants with an eighteen amino acid peptide amended to their amino terminus. Complementation analysis has also been used to determine how the extra residues added to protein termini influence where AK tolerates permutation. In all variants tested to date, similar complementation strength has been observed in the presence of the different peptide tags amended by our different transposons, suggesting that protein stability does not contribute to our library trends. Instead, we hypothesize that translation initiation efficiency varies among our clones, which may pose a fundamental expression challenge when building libraries of circularly permuted proteins. Calculations using a thermodynamic model for translation initiation support this idea by showing that permuted AK with high calculated expression retained function with a higher frequency than variants chosen at random from each library. To date, our findings expand the library sequence diversity that can be accessed using transposon mutagenesis, and they show that libraries created using different transposons should be complementary in discovering functional permuted proteins. However, these findings illustrate a fundamental expression challenge when evolving proteins by altering their gene architecture through permutation.