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Non-natural amino acid-charged tRNAs for site directed protein functionalization.

CloverDirect™ is a line of products from Protein Express that permit targeted incorporation of non-natural amino acids into proteins expressed in cell-free coupled transcription/translation systems based on cell lysates or purified components. Each product comprises an aminoacyl tRNA (either UAG [amber] or CGGG) charged with a specific unnatural amino acid. To introduce unnatural amino acids at user-defined positions, templates are designed with appropriately placed UAG (amber) and/or CGGG codons. User-designed templates can be either mRNA, linear PCR products or expression plasmids.

Protein Express also offers linear template-generating PCR kits and expression vectors that encode a specialized N-terminal peptide (ProX tag) designed for large unnatural amino acids whose efficient incorporation requires precise N-terminal positioning (codon 9) and a specific codon context (flanked by AAC and TCT) (1). Originally developed for the 4 base CGGG codon, the ProX tag can also function to promote N-terminal incorporation of unnatural amino acids using the amber codon (2).

Key references for ProX tag

  1. Abe, R., Shiraga, K., Ebisu, S., Takagi, H. & Hohsaka, T. Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons. J. Biosci. Bioeng. 110, 32–38 (2010).
  2. Tada, S. et al. Genetic PEGylation. PLoS ONE 7, e49235–6 (2012).

CloverDirect™ offers off-the-shelf UAG or CGGG aminoacyl tRNAs charged with non-natural amino acids that permit:

  1. Fluorescent-labeling
  2. Biotin-labeling
  3. PEGylation
  4. Chemical crosslinking
  5. Photo-regulation of a variety of biological processes, including enzyme, receptor, and ion channel activity

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