Site-Directed Unnatural Mutagenesis, PEG4-AF (CGGG), CloverDirect

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PRX

Clover Direct ™ tRNA Reagents for Site-Directed Protein Functionalization are designed to permit the incorporation of unnatural amino acids at defined positions within proteins using cell-free coupled transcription/translation systems, including PUREfrex® , PURESYSTEM® and The RYTS Kit. The incorporation positions of unnatural amino acids are defined by selectively introducing UAG amber or CGGG four-base codons into translation templates. For template construction, we recommend Protein Express Linear Template PCR kits (available in two forms: His-tag only or ProX-tag + His-tag) or expression plasmids from Protein Express (pROX-FL92.1: ProX-tag + His-tag) or BioComber (pURE1-4: no tags). The ProX tag is a special N-terminal peptide designed for the incorporation of certain large unnatural amino acids that require precise N-terminal positioning (codon 9) and a specific codon context (flanked by AAC and TCT). Codon 9 of the ProX tag can be varied between UAG, CGGG or TTT (as a control). During cell-free translation, unnatural amino acid-charged aminoacyl-tRNAs recognize either UAG amber or CGGG codons. If a UAG codon is recognized by release factor 1 (one of three termination factors), protein synthesis will be terminated. If the first three nucleotides of CGGG are recognized as a triplet codon by Arg-tRNA, the reading frame shifts to +1 and a downstream stop codon will terminate protein synthesis. Therefore, full-length translation products will contain unnatural amino acids at 100% efficiency.
CloverDirect™ offers off-the-shelf UAG or CGGG aminoacyl tRNAs charged with unnatural 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

In addition, custom services for the expression of proteins with unnatural amino acids are available upon request.