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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).
Fluoresence Labeling
| Product Name | Size | Catalog Number |
| CloverDirect™ CR110-X-AF (amber) |
300UL | PRX-CLD1001 |
| 5*300UL | PRX-CLD2001 | |
| CloverDirect™ CR110-X-AF (CGGG) | 5*300UL | PRX-CLD2002 |
| CloverDirect™ HiLyte Fluor 488-AF (amber) |
300UL | PRX-CLD01 |
| 5*300UL | PRX-CLD05 | |
| CloverDirect™ HiLyte Fluor 488-AF (CGGG) | 5*300UL | PRX-CLD2004 |
| CloverDirect™ TAMRA-C6-AF (amber) |
300UL | PRX-CLD02 |
| 5*300UL | PRX-CLD06 | |
| CloverDirect™ ATTO633-AF (amber) |
300UL | PRX-CLD03 |
| 5*300UL | PRX-CLD07 | |
| CloverDirect™ ATTO633-AF (CGGG) | 5*300UL | PRX-CLD2008 |
| CloverDirect™ ATTO655-X-AF (amber) |
300UL | PRX-CLD1009 |
| 5*300UL | PRX-CLD2009 | |
| CloverDirect™ ATTO655-X-AF (CGGG) |
300UL | PRX-CLD1010 |
| 5*300UL |
Biotin Labeling
| Product Name | Size | Catalog Number |
| CloverDirect™ Biotin-AF (amber) | 5*300UL | PRX-CLD2101 |
| CloverDirect™ Biotin-AF (CGGG) | 5*300UL | PRX-CLD2102 |
| CloverDirect™ Biotin-X-AF (amber) | 5*300UL | PRX-CLD2103 |
| CloverDirect™ Biotin-X-AF (CGGG) | 5*300UL | PRX-CLD2104 |
| CloverDirect™ Biotin-XX-AF (amber) |
300UL | PRX-CLD04 |
| 5*300UL | PRX-CLD08 | |
| CloverDirect™ Biotin-XX-AF (CGGG) | 5*300UL | PRX-CLD2106 |
Post-Translational Modification
| Product Name | Size | Catalog Number |
| CloverDirect™ Tyr(PO3H2) (amber) | 5*300UL | PRX-CLD2201 |
| CloverDirect™ Tyr(PO3H2) (CGGG) | 5*300UL | PRX-CLD2202 |
| CloverDirect™ Lys(Me) (amber) | 5*300UL | PRX-CLD2203 |
| CloverDirect™ Lys(Me) (CGGG) | 5*300UL | PRX-CLD2204 |
| CloverDirect™ Lys(Me2) (amber) | 5*300UL | PRX-CLD2205 |
| CloverDirect™ Lys(Me2) (CGGG) | 5*300UL | PRX-CLD2206 |
| CloverDirect™ Lys(Ac) (amber) | 5*300UL | PRX-CLD2207 |
| CloverDirect™ Lys(Ac) (CGGG) | 5*300UL | PRX-CLD2208 |
Unnatural Mutagenesis
| Product Name | Size | Catalog Number |
| CloverDirect™ PEG4-AF (amber) | 5*300UL | PRX-CLD2301 |
| CloverDirect™ PEG4-AF (CGGG) | 5*300UL | PRX-CLD2302 |
| CloverDirect™ PEG8-AF (amber) | 5*300UL | PRX-CLD2303 |
| CloverDirect™ PEG8-AF (CGGG) | 5*300UL | PRX-CLD2304 |
| CloverDirect™ PEG12-AF (amber) | 5*300UL | PRX-CLD2305 |
| CloverDirect™ PEG12-AF (CGGG) | 5*300UL | PRX-CLD2306 |
| CloverDirect™ BPA (amber) | 5*300UL | PRX-CLD2321 |
| CloverDirect™ BPA (CGGG) | 5*300UL | PRX-CLD2322 |
| CloverDirect™ AcPhe (amber) | 5*300UL | PRX-CLD2323 |
| CloverDirect™ AcPhe (CGGG) | 5*300UL | PRX-CLD2324 |
| CloverDirect™ AzoAla (amber) | 5*300UL | PRX-CLD2331 |
| CloverDirect™ AzoAla (CGGG) | 5*300UL | PRX-CLD2332 |
Key references for ProX tag
- 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).
- 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:
- Fluorescent-labeling
- Biotin-labeling
- PEGylation
- Chemical crosslinking
- Photo-regulation of a variety of biological processes, including enzyme, receptor, and ion channel activity