科研之友
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摘要
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 similar to 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.
- 出版日期2013-3
