Protein Intein

Harnessing protein splicing, researchers now have the ability to generate recombinant protein a-thioesters through the thiolysis of an appropriately mutated protein-intein fusion. In principle, this means that synthetic and recombinant building blocks can be fused in a semisynthetic version of NCL. Such an approach was first reported in 1998 and has been named expressed protein ligation [8],... 

D.W. (2005) Novel and economical purification of recombinant proteins Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci., 14, 1387-1395. 

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+.

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.

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. 

Fig. 1.4 A I ntein-mediated protein ligation (IPL) (also called expressed protein ligation EPL). B The chemical mechanism of protein cyclization by the IPL/EPL approach. HAC denotes the sequence of the active site of the intein, e.g. His-Ala-Cys. CBD stands for chitin-binding domain.

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

Fig. 1.5 A Natural protein splicing, B Trans splicing with a split intein. The two fragments can be prepared separately and reassembled in vitro to form an active intein domain for protein ligation. C Central segment labeling using two different split inteins.

---