Intein Mediated Protein Ligation IPL

Unlike other affinity fusion systems that mainly serve to simplify protein purification, the intein fusion systems can be modulated to perform numerous other tasks, one of which is intein-mediated protein ligation (IPL; Evans et al. 1998; Evans and Xu 1999) or expressed protein ligation (Muir et al. 1998; Severinov and Muir 1998). The IPL technique has been used for incorporation of non-coded amino acids into a protein sequence, segmental labeling of proteins for NMR analysis, addition of fluorescent probes to create biosensors, and synthesis of cytotoxic proteins (Cotton and Muir 1999,2000; Otomo et al. 1999; Xu et al. 1999). These applications utilize essentially the same purification protocols. By altering cleavage conditions, the intein fusion systems can generate a protein with a reactive C-terminal thioester or an N-terminal cysteine. The facile generation of thioester-tagged recombinant proteins has greatly expanded the utility of native chemical ligation (Dawson et al. 1994; Tam et al. 1995). The latter method was developed for fusion of two synthetic peptides, one possessing a C-terminal thioester and the other possessing an N-terminal cysteine. The ability of IPL to generate large recombinant protein substrates under mild conditions circumvents the problem associated with synthetic peptide intermediates which are usually limited to less than 100 amino acids. Further advance of the IPL method has been achieved by employing the intein C-terminal cleavage system to generate proteins with an N-terminal cysteine (Evans et al. 1999b; Mathys et al. 1999). The utility of various intein fusion systems has contributed to an alternate approach in backbone cyclization of large recombinant proteins as well as peptides. Cyclic proteins/ peptides can be generated by creating an N-terminal cysteine residue on the same protein possessing a C-terminal thioester (Evans et al. 1999a; Iwai and Pluckthun 1999; Xu et al. 1999). The following sections describe some of our new applications of IPL technology.

4.1 Intein-Mediated Peptide Array (IPA)

Peptides are widely used to generate antibodies, define antibody epitopes and determine substrate specificities of protein modification enzymes such as protein kinases and phosphatases. Binding of peptides to commonly used membranes such as nitrocellulose is ineffective and variable, resulting in low sensitivity and inconsistency. A novel method for making arrays of synthetic peptides has been developed by ligating the peptide substrates to a carrier protein which is generated by the intein fusion system and has a high binding affinity for nitrocellulose (Sun et al. 2004). For example, when the synthetic peptide antigens were ligated to a thioester-tagged methylase from

Haemophilus haemolyticus (M.Hha, 39 kDa) or the paramyosinASal fragment (27 kDa) from Dirofilaria immitis, the use of the ligation products resulted in improved retention of peptides and an increase in sensitivity of up to 104-fold in immunoassay and epitope scanning experiments (Fig. 6). Because the carrier protein contains one peptide reactive site and dominates the binding of ligated products, the amount of peptides arrayed onto the membranes can be a

HA tag HA tag HA tag HA tag HA tag HA tag HA tag HA tag HA tag b

PI P2 P3 P4 P5 P6 P7 P9 P9 nmol

Fig. 6. Alanine scan of a hemagglutinin (HA) epitope by the intein-mediated peptide array (IPA) method (Sun et al. 2004). a An alanine-scanning HA peptide library was synthesized with an N-terminal cysteine. P9 contains the wild-type sequence corresponding to residues 98 to 106 of HA protein (YPYDVPDYA). Each of the other eight peptides carries a single substitution with an alanine residue. Each peptide (0.5 mM) was ligated to thioester-tagged paramyosin (25 ^M) at 4 °C overnight, b The ligated products (rows 1-4) and the unligated peptides (rows 5-8) were threefold serially diluted in phosphate-buffered saline (PBS) and arrayed on a 0.45-^m nitrocellulose membrane. Immunoblotting was performed with an-ti-HA monoclonal antibody (Zymed Laboratories) and detection was with the PhototopeHRP Western blot detection system (Cell Signaling Technology). The amount of total peptide present in each sample is indicated on the right side. The data indicated that the residues mutated in PI, P2, P3, and P4 are essential for antibody recognition and the unligated peptides did not generate a detectable signal

P5 CAGAG YPYD0PDYA

P7 CAGAG YPYDVP[X]YA

P9 CAGAG YPYDVPDYA

effectively normalized. This IPL generated peptide array (IPA) approach permits synthetic peptides to be uniformly arrayed onto commonly used membranes (i.e., nitrocellulose, Nylon, and PVDF) thereby making it convenient and economical to produce peptide arrays in a research laboratory.

4.2 Kinase Assays Using Carrier Protein-Peptide Substrates

Another example of the application of IPL is the generation of improved peptide substrates for kinase assays and subsequent Western blot analysis and array (Fig. 7). Synthetic peptide substrates have become a convenient tool to determine kinase specificities and to conduct mutational analysis but they are not suitable for Western blot analysis due to their small size. IPL allows for ef-

Intein Protein Ligation

Fig. 7. A schematic illustration of using IPL to generate peptide substrates. A carrier protein possessing a cysteine-reactive C-terminal thioester is released following intein-mediated cleavage by the addition of 2-mercaptoethanesulfonic acid (MESNA). Ligation of this carrier protein to a synthetic peptide containing an N-terminal cysteine as well as a potential phosphorylation site (tyrosine or Y) results in a native peptide bond between the two reacting species. The ligated carrier protein-peptide product serves as substrate for kinase assays and subsequent Western blot analysis with a phospho-tyrosine antibody (Xu et al. 2004). Phosphorylation of the tyrosine residue in the peptide sequence (indicated by a circled P) results in a positive signal

Fig. 7. A schematic illustration of using IPL to generate peptide substrates. A carrier protein possessing a cysteine-reactive C-terminal thioester is released following intein-mediated cleavage by the addition of 2-mercaptoethanesulfonic acid (MESNA). Ligation of this carrier protein to a synthetic peptide containing an N-terminal cysteine as well as a potential phosphorylation site (tyrosine or Y) results in a native peptide bond between the two reacting species. The ligated carrier protein-peptide product serves as substrate for kinase assays and subsequent Western blot analysis with a phospho-tyrosine antibody (Xu et al. 2004). Phosphorylation of the tyrosine residue in the peptide sequence (indicated by a circled P) results in a positive signal ficient ligation of a synthetic peptide substrate with an N-terminal cysteine residue to an intein-generated carrier protein (with a size range of 20-40 kDa; Evans and Xu 1999). A distinct advantage of this procedure is that since each carrier protein molecule ligates only one peptide, the resulting ligation product migrates as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The peptide substrates may contain a desired amino acid substitution or chemical modification. This design has led to the mutational analysis of the peptide substrates derived from human cyclin-de-pendent kinase, Cdc2, that contains a phosphorylation site for human c-Src protein tyrosine kinase (Xu et al. 2004). A peptide possessing a phosphorylation site of interest is synthesized with an amino-terminal cysteine residue. The peptide is ligated to the cysteine-reactive carboxyl terminus of a carrier protein via a peptide bond. Following assays with the Src kinase, protein phosphorylation is subsequently examined by Western blot analysis with a phos-pho-specific antibody that recognizes the phosphorylated tyrosine epitopes.

4.3 Purification of Peptide-Specific Antibody

Affinity purification of a peptide-specific antibody relies on conjugation of peptide antigens to agarose or other matrices. This procedure is often time-consuming and generates chemical waste. By taking advantage of the IPL technique and the high affinity of the chitin-binding domain (CBD) for chitin, a method has been developed to produce a peptide affinity column (Sun et al. 2003). A reactive thioester is first generated at the C-terminus of the CBD from the chitinase Al of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were subsequently ligated to the chitin-bound CBD tag. It has been shown that the resulting peptide columns are highly specific and efficient in affinity purification of antibodies from animal sera.

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