Encoded peptide tags

Evans, W.J., Allen, N.T., and Ziller, J.W. (2002) Protein affinity labeling mediated by genetically encoded peptide tags. Angewandte Chemie International Edition, 41, 359. 

Peptides have also been used as tags (Figure 6.12) in a similar manner to DNA oligo nucleotides. The sequence of amino acids in the encoding peptide is determined using the Edman sequencing method. This amino acid sequence is used to determine the history of the formation and hence the structure of the product found on that bead. 

The encoding tags are halophenoxy derivatives of aliphatic alcohols and are attached to either a photolabile linker as a carbonate or an oxidatively cleavable linker as an ether. The photolabile linker/tag complex is especially well suited to encode peptide libraries through attachment to approximately 1% of the amino groups of each amino acid synthon at eachstage of the synthesis. For structure determination, the tags are released by photolysis at 360 nM. 

Fig. 4. Role of the stop codon and lOSa-RNA in E. coli translation. (A) When a stop codon is encountered, a complex of two release factors, RF-1 and RF-3 or RF-2 and RF-3, binds instead of the tRNA. The release factor RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. The binding of the release factor complex results in hydrolysis of the peptidyl-tRNA and release of the peptide. (B) The role of lOSa-RNA. If truncated mRNA without a stop codon is translated in E. coli, the ribosome stops at the end of the mRNA. lOSa-RNA can then bind to the ribosomal A site and lOSa-RNA can act as tRNA by transferring an alanine to the truncated protein. Subsequently, lOSa-RNA acts as mRNA and a peptide tag with the indicated sequence is added to the truncated protein. lOSa-RNA encodes a stop codon and therefore the protein is released and then degraded by proteases specifically recognizing this C-terminal tag.

Yin J, Straight PD, McLoughlin SM, Zhou Z, Lin AJ, Golan DE, Kelleher NL, Kolter R, Walsh CT. Genetically encoded short peptide tag for versatile protein labehng by Sfp phosphopantetheinyl transferase. Proc. Natl. Sci. U.S.A. 2005 102 15815-15820. 

Zhou, Z., Cironi, P., Lin, A. J., Xu, Y., Hrvatin, S., Golan, D. E., etal. (2007). Genetically encoded short peptide tags for orthogonal protein labeling by S and AcpS phosphopantetheinyl transferases. CS Chemical Biology, 2, 337—346. 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

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