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

Abstract

We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the 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 N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-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 N(3)-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 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.

Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate

“…The breakthroughs in this study were ( a ) the use of a tac promoter and ( b ) the combination of a novel culture system and auto-induction culture medium. The former and the latter were shown to be useful in previous reports by Ikeda-Boku et al [ 17 ] and Li et al [ 18 ], respectively. When the protein was expressed from a tac promoter at 37 °C, the ratio of soluble to insoluble recombinant oANG increased significantly (Fig.…”

Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA‐modifying enzyme, archaeal tRNA‐guanine transglycosylase

“…The pTPP_SerW vector for constitutive expression of E. coli tRNA Ser (GGA) has the tRNA Ser (GGA) gene (serW) inserted into the pTPP vector multicloning site (Ikeda-Boku et al 2013). A series of single-gene-disrupted E. coli strains and a host strain harboring pTPP_SerW were cultured at 37°C in 3 L of Luria-Bertani medium containing 20 lg/mL kanamycin and chloramphenicol.…”

Rewriting the Genetic Code

“…O -phosphoserine (Sep), O -phosphotyrosine (pTyr; 32), and N ε -acetyllysine (103) are all common posttranslational modifications. These compatibilities with cellular systems, in turn, facilitate degradation and metabolism of free ncAAs, ncAA residues in proteins, and ncAA moieties of ncAA-tRNAs (41, 49, 116) (Figure 1 b ). Some ncAAs scramble cellular processes (118).…”

“…Furthermore, elimination of tyrosine/aspartate aminotransferases prevented conversion of p -hydroxy-L-phenyllactic acid to tyrosine (41). Depletion of two reductase genes ( trxB and gor ) prevented the reduction of azido-tyrosine to amino-tyrosine (49). A mutation in the arginine repressor ArgR(L70P) eliminated the toxicity of homoarginine in the E. coli B strain (96, 118).…”