Mini-intein

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... 

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... 

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. 

The modified Mth RIRl, Mxe GyrA, and Ssp DnaB mini-inteins have been recently applied to the isolation of proteins with an N-terminal cysteine residues (29,30). These inteins undergo temperature- and pH-dependent C-termi-nal cleavage when the N-terminal cysteine residue of the intein is substituted with alanine (Table 2). The target protein is recombinantly expressed as a fusion protein with the C-terminal intein tag (31) (Fig. 3B). After intein splicing the protein that possesses N-terminal cysteine is generated. Moreover, such a protein can be obtained by total chemical synthesis and different chemical labels or non-canonical amino acids can be site-specifically incorporated into the sequence. 

Mathys, S., Evans, T. C., Chute, I. C., et al. (1999) Characterization of a self-sphcing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements facile production of protein building blocks for protein ligation. Gene 231, 1—13. 

Fig. 2 Structural model of Mycobacterium tuberculosis recA mini intein (PDB 2IMZ) after the splicing reaction. The N-terminal Cys is in close proximity of the C-terminal succinimide...

Inteins have several signature motifs (Fig. 1 and Table I). Blocks A, B, F, and also known as Blocks Nl, N3, C2, and Cl, respectively, are present in all inteins. Block A begins with the first residue of the intein and Block B is usually 60-100 amino acids from the start of the intein. Block F closely precedes Block G, which includes the end of the intein and the first residue of the carboxy-extein. Blocks C, D, E, and H (also known as Blocks ENl-4 ) are sometimes present between Blocks B and F and include the signature motifs of one class of endonuclease, termed the dodecapeptide (DOD) or LAGLI-DADG family of homing endonucleases (see below). Mini-inteins (<200 aa) have a small linker in place of the homing endonuclease domain. 

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