Non-canonical amino acids in protein engineering

Link, A. James; Mock, Marissa; Tirrell, David A.

doi:10.1016/j.copbio.2003.10.011

Cited by 385 publications

(244 citation statements)

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“…Recent efforts aimed at producing proteins with specifically modified residues have provided many examples of how the genetic code of Bacteria and Eucarya can be modulated by laboratory mutation of canonical aminoacyl-tRNA synthetase and cognate tRNA pairs to accept unnatural amino acids (18)(19)(20). In one case, an additional mutant biosynthetic enzyme enabled autogenous biosynthesis and genetic encoding of aminophenylalanine in E. coli (26).…”

Section: Discussionmentioning

confidence: 99%

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A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Longstaff

Larue

Faust

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding tRNA Pyl that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Archaea but also in a distantly related Gram-positive Bacterium, indicating past horizontal gene transfer of all five genes. Here we show that lateral transfer of pylTSBCD introduces biosynthesis and genetic encoding of pyrrolysine into a naïve organism. PylS-based assays demonstrated that pyrrolysine was biosynthesized in Escherichia coli expressing pylBCD from Methanosarcina acetivorans. Production of pyrrolysine did not require tRNA Pyl or PylS. However, when pylTSBCD were coexpressed with mtmB1, encoding the methanogen monomethylamine methyltransferase, UAG was translated as pyrrolysine to produce recombinant monomethylamine methyltransferase. Expression of pylTSBCD also suppressed an amber codon introduced into the E. coli uidA gene. Strains lacking one of the pylBCD genes did not produce pyrrolysine or translate UAG as pyrrolysine. These results indicated that pylBCD gene products biosynthesize pyrrolysine using metabolites common to Bacteria and Archaea and, furthermore, that the pyl gene cluster represents a ''genetic code expansion cassette,'' previously unprecedented in natural organisms, whose transfer allows an existing codon to be translated as a novel endogenously synthesized free amino acid. Analogous cassettes may have served similar functions for other amino acids during the evolutionary expansion of the canonical genetic code.T ranslated in-frame amber (TAG/UAG) codons in the genes encoding methylamine methyltransferases from Methanosarcina barkeri (1-3) were early clues to the existence of pyrrolysine as the 22nd genetically encoded amino acid from nature. Crystallography of MtmB1, the monomethylamine methyltransferase, demonstrated that the UAG codon of the encoding mtmB1 gene corresponds to pyrrolysine, whose structure was proposed as lysine with N in amide linkage with the D-isomer of 4-methyl-pyrroline-5-carboxylate (4, 5). This structure is consistent with the UAG-encoded residue's mass in three distinct methylamine methyltransferases (6).Near the mtmB1 gene in Methanosarcina spp. are the pyl genes (ref. 7; Fig. 1). Two of these genes are known to underlie the genetic encoding of pyrrolysine. The pylT encodes tRNA Pyl , whose CUA anticodon complements the UAG codon corresponding to pyrrolysine. Deletion of the pyl gene promoter and pylT leads to loss of pyrrolysine-dependent monomethylamine methyltransferase and methylamine metabolism (8). The pylS gene encodes the pyrrolysyl-tRNA synthetase, PylS, which charges tRNA Pyl with chemically synthesized pyrrolysine and further carries out a pyrrolysine-dependent ATP:pyrophosphate exchange reaction (5, 9-11)...

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Section: Discussionmentioning

confidence: 99%

“…be a powerful avenue to artificially manipulating genetic codes (18)(19)(20). However, transfer of a ''genetic code expansion cassette'' would be unprecedented in natural gene pools.…”

mentioning

confidence: 99%

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Longstaff

Larue

Faust

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…An overview of the most important technologies that permit the covalent incorporation of unnatural molecules into proteins is illustrated in Figure 1 and discussed here in the next pages. [6][7][8][9][10][11] Isolation of a particular protein among a myriad of other proteins and molecules found inside cells is not a trivial task either by laboratory synthesis or cell population purification. For the past 20 years, this task has been enormously facilitated by the development of biotechnological methods involving the recombinant DNA-based expression of proteins in genetically engineered cells.…”

Section: In the World Of Protein Sciencementioning

confidence: 99%

Diels–Alder Ligation of Peptides and Proteins

Araújo

Palomo

Cramer

et al. 2006

Chemistry A European J

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“…Whereas the global misaminoacylation strategy aims to change the overall properties of the proteins, the site-directed incorporation strategy allows more subtle and specialized changes in the proteins (Link et al, 2003). It can be speculated that a combination of these strategies could be used to produce extensively modified recombinant proteins for pharmaceutical and other applications.…”

Section: Site-directed Incorporation Of Unnatural Amino Acidsmentioning

confidence: 99%