Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Sep 16;94(19):10092-7.
doi: 10.1073/pnas.94.19.10092.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

D R Liu  1 , T J Magliery, M Pastrnak, P G Schultz
Affiliations

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

D R Liu et al. Proc Natl Acad Sci U S A. .

Abstract

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sequence of tRNA2Gln and the base changes converting tRNA2Gln to the orthogonal-tRNA (O-tRNA). The interactions between knob 1, knob 2, and knob 3 in the tRNA with residues Asp-235, Glu-323, and Gln-13 in GlnRS are also depicted (19).
Figure 3
Figure 3
In vivo suppression of lamB amber mutants by the O-tRNA and mutant GlnRS enzymes. Crude cell lysates were subjected to SDS/PAGE and the resulting proteins were visualized by Western blot using anti-LamB polyclonal antibodies. Lanes: 1, XL-1 blue cells (a lamB positive strain); 2, GS-20 cells (a strain lacking the lamB gene); 3–18, GS-20 cells transformed with pAC123LamB harboring either the N72Am or the D200Am lamB mutant gene as indicated; 3–6, pBR322 transformants; 7–10, pBRGlnRS clone 5/3 transformants; 11–14, pBRGlnRS clone 6/4 transformants; 15–18, pBRGlnRS clone 7/6 transformants. Induction of mutant LamB expression with arabinose is indicated above each lane.
Figure 2
Figure 2
Flow chart describing the history of each GlnRS library. Thick arrows denote a round of DNA shuffling and selection on the media indicated next to each arrow. LMM, lactose minimal media. Thin arrows indicate that a small number of characterized GlnRS genes were used to begin a new round of DNA shuffling and selection. Mutagenic oligonucleotides, when added to a reassembly reaction during DNA shuffling, are listed in boldface type.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mendel D, Cornish V W, Schultz P G. Annu Rev Biophys Biomol Struct. 1995;24:435–462. - PubMed
    1. Cupples C G, Miller J H. Genetics. 1988;120:637–644. - PMC - PubMed
    1. Cornish V W, Hahn K M, Schultz P G. J Am Chem Soc. 1996;118:8150–8151.
    1. Yamao F, Inokuchi H, Ozeki H. Jpn J Genet. 1988;63:237–249. - PubMed
    1. Thomann H-U, Ibba M, Hong K-W, Söll D. Bio/Technology. 1996;14:50–55. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources