A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli

We developed an efficient method for introduction of 3-azidotyrosine (N₃-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable forthe constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(CUA), and made an orthogonal tRNA(CUA) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N₃-Y was selected. We then expressed rat calmodulin (CaM) containing N₃-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N₃-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N₃-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/ tRNA(CUA) gene. Although the yields of full-length CaM increased ∼3-fold, the ratio of N₃-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N ₃-Y at the pre-determined site. Finally, we obtained up to 2mg of CaM containing N₃-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.