A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli.
J Biochem
; 153(3): 317-26, 2013 Mar.
Article
en En
| MEDLINE
| ID: mdl-23316081
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tirosina
/
Proteínas Recombinantes
/
Calmodulina
/
Escherichia coli K12
Límite:
Animals
Idioma:
En
Revista:
J Biochem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Reino Unido