Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

Immobilized metal-ion affinity (IMA) adsorption is a collective term that is used to include all kinds of adsorptions where the metal ion serves as the characteristic and most essential part of adsorption center. Of all the IMA techniques, immobilized metal-affinity chromatography (IMAC) has been gaining popularity as the choice of purification technique for proteins. IMAC represents a separation technique that is primarily useful for proteins with natural surface exposed-histidine residues and for recombinant proteins with engineered histidine tag. This review also gives insight into other nonchromatographic applications of IMA adsorption such as immobilized metal-ion affinity gel electrophoresis (IMAGE), immobilized metal-ion affinity capillary electrophoresis (IMACE), and immobilized metal-ion affinity partitioning (IMAP).

The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.

Characterization of an Exo-β-1,3-Galactanase from Clostridium thermocellum

ABSTRACT A gene encoding an exo-β-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-β-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50�C and catalyzes hydrolysis only of β-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from β-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a β-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-β-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including β-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a β-1,3-galactobiose, β-1,4-galactosyl glucose, or β-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward β-1,3-galactan and α-l-arabinofuranosidase-treated AGP.