Inteins

Rekalitis, G. V, and H. D. Spriggs. Proceedings of the First Inteinational Conference on Foundations of Computer-Aided Operations. Elsevier Science Publishers, Inc., New York (1987). 

Perler FB. InBase the Intein Database. Nucleic Acids Res 2002 30 383-384. 

Comprehensive reviews published by Snodderly and Beatty et al. " explore the evidence for a protective fnnction by the macular pigment against age-related macnlar diseases and the mechanisms by which it might act. The antioxidant properties of Intein and zeaxanthin recently reviewed by Young and Lowe may rednce the degree to which oxidative damage promotes these diseases. Otherwise, becanse these... 

In fasting hnman sernm, the hydrocarbon carotenes (P-carotene and lycopene) are fonnd primarily in LDL, while the xanthophylls (Intein, zeaxanthin, and P-cryptox-anthin) are more evenly distribnted between LDLs and HDLs. As mentioned earlier and contrary to the carotenes, the xanthophylls are primarily located at the surfaces of lipoprotein particles, making them more likely to exchange between plasma lipoproteins. This hypothesis may explain their eqnal distribntion (or apparent equilibrinm) between LDLs and HDLs. 

Britton, G., UV/visible spectroscopy, in Carotenoids Spectroscopy, IB, Britton, G., Liaaen-Jensen, S., and Pfander, H., Eds., Birkhanser, Basel, 1995, 13. Melendez-Martinez, A.J. et ah. Identification of isolntein (Intein epoxide) as cis-antheraxanthin in orange juice, J. Agric. Food Chem., 53, 9369, 2005. 

Figure 17.13 Expressed proteins containing a thioester intein tag can be specifically modified using a cysteine-alkyne derivative by transthioesterification followed by an internal S - N shift.

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

Figure 17.26 The native process leading to intein excision and ligation of extein fragments involves a sequence of reactions involving transthioesterification, cleavage of the intein fragment, and aS->N shift, which ligates the two extein peptides together via an amide bond.

Fusion vectors are available that combine a recombinant protein with a mutant mini intein segment (not containing an endonuclease domain) and followed by a chitin binding domain (CBD Zhang et al., 2001). These mutants typically also have an alanine substitution that replaces the cysteine or serine/threonine usually found on the C-extein splice junction. Alanine... 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

EPL extends the applicability of native chemical ligation to recombinantly produced proteins using the mutant mini intein vector system. Proteins being expressed using this method will... 

Fusion protein containing the expressed protein, the mutant intein, and the CBD... 

Intein cleavage and release of expressed protein with N-terminal Cys... 

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Figure 17.29 An expressed protein containing a mutant intein segment can undergo self cleavage to form an N-terminal cysteine residue, which then can be reacted with a thioester probe to label specifically the protein via an amide bond.

Perler, F.B. (2000) InBase, the intein database. Nucleic Acids Res. 28, 344-345. 

Zhang, A., Gonzalez, S.M., Cantor, E.J., and Chong, S. (2001) Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins. Gene 275, 241-252. 

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