CH391L/S12/Unnatural Amino Acids

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#A selectable (Ab resistance) marker with at least one in-frame Amber codon
#A selectable (Ab resistance) marker with at least one in-frame Amber codon
#Exogenously supplied unnatural amino acid
#Exogenously supplied unnatural amino acid
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The primary challenge to overcome in the development of these systems is the fulfilling the criterion of aaRS/tRNA orthogonality and aminoacylation specificity. The best starting point for this goal is to import an aaRS/tRNA pair from a different domain of life. The orthogonality of the pair must then be improved by rounds of positive selection to obtain individuals that successfully incorporate the unnatural amino acid of choice and negative rounds of selection to ensure that canonical amino acids are not incorporated at the Amber codon. This process is diagrammed at left.
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The primary challenge to overcome in the development of these systems is the fulfilling the criterion of aaRS/tRNA orthogonality and aminoacylation specificity. The best starting point for this goal is to import an aaRS/tRNA pair from a different domain of life. The orthogonality of the pair must then be improved by rounds of positive selection to obtain individuals that successfully incorporate the unnatural amino acid of choice and negative rounds of selection to ensure that canonical amino acids are not incorporated at the Amber codon. This process is diagrammed below.
[[Image:UAA_selection.png|thumb|left|250px|Positive and negative rounds of selection for UAA incorporation <cite>Liu2010</cite>]]
[[Image:UAA_selection.png|thumb|left|250px|Positive and negative rounds of selection for UAA incorporation <cite>Liu2010</cite>]]

Revision as of 20:11, 22 March 2012

Contents

Unnatural Amino Acids

Introduction

The genetic code for the translation of RNA into protein is one of the most ancient and universal innovations in the evolution of life on earth. Nearly all life forms use the same redundant code for the incorporation of the 20 canonical amino acids into proteins. In two unique exceptions, selenocysteine and pyrrolysine, stop codons have been retooled to code for a 21st amino acid. [1][2] Expansion of the genetic code to include noncanonical, or unnatural amino acids (UAAs) holds promise for improving and diversifying protein function, generating proteins that normally would require postranslational modification, and the study of the genetic code itself. Technology for the creation of proteins bearing UAAs has progressed steadily over the last ~30 years, including both in vitro and in vivo methods.

In vitro Synthesis

Schematic presentation of the principles of solid-phase peptide synthesis[3]
Schematic presentation of the principles of solid-phase peptide synthesis[3]
Principles of chemical ligation[3]
Principles of chemical ligation[3]
A method of native chemical ligation
A method of native chemical ligation

Solid-phase Synthesis and Chemical Ligation

Solid-phase peptide synthesis (SPPS) was developed by Bruce Merrifield in the early '60s, for which he received the Nobel Prize in 1984. In this method, the C-terminal amino acid is anchored via a linker to an insoluble support. Both the N-terminus and the side-chain are protected from reaction. The N-terminus is typically protected by Boc or Fmoc groups, and the side-chains can be protected by a variety of groups. In the first step, the N-terminus is deprotected. The next desired amino acid in the chain is added to the column. This process of deprotection and addition is repeated until the chains is completed. The side-chains are then deprotected, and the completed peptide is eluted from the column. This method is useful for producing peptides of up to ~50 amino acids, and noncanonical amino acids may be readily incorporated.

Native chemical ligation is used to produce larger peptides and full proteins. This method requires uniquely reactive functionalities incorporated into each peptide at the N- and C-terminus, and allows the use of unprotected peptide segments. In one method of native chemical ligation, the thiolate group of an N-terminal cysteine residue peptide attacks the C-terminal thioester of a second unprotected peptide. This reversible transthioesterification step is chemoselective and regioselective and leads to form a thioester intermediate. This intermediate rearranges irreversibly by an intramolecular S,N-acyl shift that results in the formation of a native peptide bond at the ligation site. This method may be repeated to make long peptides and proteins.[4]

Synthesis via Chemically Aminoacylated tRNAs

Proteins with unnatural amino acids may also be produced biosynthetically. In this technique, truncated tRNAs are enzymatically ligated to chemically aminoacylated nucleotides, effectively decoupling the identity of the tRNA from that of the attached amino acid.[5] One can then use a cell-free translation system to synthesize proteins with unnatural amino acids incorporated at the codon complementary to the tRNA used - typically an unused stop codon like Amber (UAG). [6] The protein may then be isolated and its properties analyzed.

In vivo Approaches

Amino Acid Auxotroph Substitution

Strains auxotrophic for a canonical amino acid can incorporate close structural analogs into proteins. Cells may be grown in presence of the canonical amino acid, and then removed from the growth medium and inoculated into growth medium containing none of the canonical amino acid, but an overabundance of a close structural analog. While this analog is usually not able to sustain exponential growth, nondividing cells are still viable and able to overexpress proteins containing this analog.[7]

In vivo Amber Codon Suppression

In vivo unnatural amino acid incorporation via Amber suppression. Retrieved from wang.salk.edu/
In vivo unnatural amino acid incorporation via Amber suppression. Retrieved from wang.salk.edu/

In the late 1990s and early 2000s, the Schultz group at Scripps developed the technology to generate organisms with an expanded 21 amino acid genetic code.[8, 9, 10] These systems are diagramed at right and generally consist of the following components:

  1. An orthongonal Amber stop codon (UAG) suppressor tRNA
  2. Evolved aminoacyl tRNA synthetase (aaRS) to charge specific unnatural amino acids on Amber suppressor tRNA
  3. A selectable (Ab resistance) marker with at least one in-frame Amber codon
  4. Exogenously supplied unnatural amino acid

The primary challenge to overcome in the development of these systems is the fulfilling the criterion of aaRS/tRNA orthogonality and aminoacylation specificity. The best starting point for this goal is to import an aaRS/tRNA pair from a different domain of life. The orthogonality of the pair must then be improved by rounds of positive selection to obtain individuals that successfully incorporate the unnatural amino acid of choice and negative rounds of selection to ensure that canonical amino acids are not incorporated at the Amber codon. This process is diagrammed below.

Positive and negative rounds of selection for UAA incorporation [11]
Positive and negative rounds of selection for UAA incorporation [11]



References

  1. Longstaff DG, Larue RC, Faust JE, Mahapatra A, Zhang L, Green-Church KB, and Krzycki JA. . pmid:17204561. PubMed HubMed [Longstaff2007]
  2. Böck A, Forchhammer K, Heider J, and Baron C. . pmid:1838215. PubMed HubMed [Boeck1991]
  3. Dawson PE and Kent SB. . pmid:10966479. PubMed HubMed [Dawson2000]
  4. Schnölzer M and Kent SB. . pmid:1566069. PubMed HubMed [Schnolzer1992]
  5. Hecht SM, Alford BL, Kuroda Y, and Kitano S. . pmid:248056. PubMed HubMed [Hecht1979]
  6. Noren CJ, Anthony-Cahill SJ, Griffith MC, and Schultz PG. . pmid:2649980. PubMed HubMed [Noren1989]
  7. Link AJ, Mock ML, and Tirrell DA. . pmid:14662389. PubMed HubMed [Link2003]
  8. Liu DR and Schultz PG. . pmid:10220370. PubMed HubMed [Liu1999]
  9. Wang L, Xie J, and Schultz PG. . pmid:16689635. PubMed HubMed [Wang2004]
  10. Wang L and Schultz PG. . pmid:11564556. PubMed HubMed [Wang2001]
  11. Liu CC and Schultz PG. . pmid:20307192. PubMed HubMed [Liu2010]
  12. Muir TW. . pmid:12626339. PubMed HubMed [Muir2003]
All Medline abstracts: PubMed HubMed
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