Chitin-binding domain

Fig. 18 CG15920 gene sequence and primary structure. The consensus repeat sequences are also represented. The highlighted regions correspond to the signal sequence, R R chitin-binding domain, and the elastomeric domains containing repeat motifs A and B. Reproduced from [182, 188] with permission from Elsevier, copyright Elsevier 2001, 2010...

Fig. 21 Chitin binding of 6xHis-tagged resilin with chitin-binding domain (6 H-resChBD) as compared to 6xHis-tagged resilin without chitin-binding domain (6 H-res). T total protein after affinity chromatography purification, B bound protein eluted from chitin beads, UB unbound protein. Reproduced from [187] with permission from The American Chemical Society, copyright 2009...

Fig. 10.4. (Opposite) Alignments of nematode chitinases with the active centre boxed. Identical amino acids are marked with an asterisk ( ) similar amino acids are marked with a dot (.). Conserved cysteine residues in the chitin-binding domain are printed in bold. Gene Bank accession numbers C.eleg. Chit (C. elegans), Q11174 OvL3 Chit (O. volvulus), L42021 AvL3 Chit (A viteae), U14638 B.m. Chit(B. malayi), M73689.

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... 

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.

Fig. 1.6 A IPL/EPL cyclization with the IMPACT system, B backbone cyclization using the TWIN inteins. CBD stands for chitin-binding domain. N-termini are indicated by NH2.

A full-length human csk DNA that codes for Csk, a 50-kDa protein that catalyzes the phosphorylation of a tyrosine within the C-terminal tail of Srk, was inserted in a plasmid and in-frame with a modified intein-chitin binding domain (CBD) encoding sequence, where intein is a protein-splicing element. 130 The expressed protein underwent a conversion into a thioester of the N-terminal cysteine of the intein, but the normal second step of an intein splicing did not occur. The protein was then bound to a chitin resin and washed. The resin containing the bound protein was treated with 2% benzenethiol to cleave the thioester and give the free phenyl thioester, which was immediately treated with one of the synthetic... 

Figure 4.8 The intein system as used for fusion proteins (Xu, 2000). The fusion proteins undergo self-cleavage at the upstream splice junction. The amino acids that participate directly are shown, and the remainder of the intein and target protein are drawn as boxes. Usually, the intein is conjugated to a chitin-binding domain, so purification of the fusion protein can occur by using a chitin-conjugated column. Self-cleavage can occur overnight.

Chen et al. (2008) have expressed a codon-optimized form of the CM4 peptide in E. coli. In this case, two alternative expression/purification systems were examined namely the widely used glutathione-S-transferase (GST) and a chitin-binding domain (CBD) system with associated intein splicing (New England Biolabs Inc.). The GST system failed to allow recovery of expressed fusion protein. In contrast, the CBD/intein system allowed the recovery of 110 mg/L fusion protein with a final RP-HPLC purification step yielding 2.1 mg/L of pure peptide which displayed antimicrobial activity against E. coli Ki2D3i and Salmonella. 

An early attempt to produce a recombinant human 29-amino acid antimicrobial peptide SMAP29 in tobacco was reported by Morassutti et al. (2002) who used a chitin-binding domain for purification and an associated intein cleavage system to release the peptide. Expression levels appeared to be low, with the peptide associated with plant proteins. Upon electrophoretic separation, the peptide displayed the expected immunological and biological activites. 

CAT-REL, vesicular catecholamine release CB-R, cannabinoid receptor CB1-R, CB2-R, cannabinoid receptors CBD, chitin-binding domain CBG, cortisol-binding globulin CBZ-R, central benzodiazepine receptor CCK-R, cholecystokinin receptor CDC, chrysanthemum dicarboxylic acid CDK, cell division kinase, cyclin-dependent protein kinase... 

The same disposition of three planar hydrophobic aromatic residues is also adopted by CBM 5 and CBM 10, as other CBMs which have Type A function and unique protein folds. An NMR structure of the 60-residue CBM 5 of an Erwinia chrysanthemi endoglucanase revealed a ski-boot structure, with a flat hydrophobic face corresponding to the sole and heel of the ski-boot. This hydrophobic face contained the three planar hydrophobic residues (two tyrosines and a tryptophan).Also using a CBM 5, but as a chitin binding domain, is the chitinase B of Serratia marcescens (the catalytic domain being GH 18). ... 

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