Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form

Abstract

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sariwus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; 11, a 109 amino acid copper-binding domain; 111, a 26 amino acid hydroxyproline-and serine-rich peptide characteristic of motifs found in the extensin family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain 11), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus verniciferu, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline-and serine-rich domain I11 suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Plantacyanin Plays a Role in Reproduction in Arabidopsis

“…Expressed protein was purified on a Ni-NTA affinity column (Qiagen) following the manufacturer's instructions. Before protein refolding, the purified protein (in 8 M urea) was incubated with the reducing reagent b-mercaptoethanol (final concentration, 10 mM) at room temperature for 1 h. A 10-fold (v/v) fast dilution was made to refold recombinant proteins by adding a refolding buffer (5 mM sodium phosphate buffer, 0.5 mM EDTA, 1 mM GSH, and 0.5 mM GSSG, pH 7.2; modified from Nersissian et al, 1996). The protein sample was kept on ice with stirring for 1 h. The refolded protein was finally dialyzed against double-distilled water at 4°C and quantified with the modified Lowry protein assay (Pierce Chemical, Rockford, IL).…”

A phytocyanin-related early nodulin-like gene, BcBCP1, cloned from Boea crassifolia enhances osmotic tolerance in transgenic tobacco

“…The four copper ligands are generally Cys, Met, and two His, with the exception of stellacyanin, where the Met is replaced by Gln (Bergaman et al, 1977). Plastocyanins and phytocyanins are two distinct subfamilies of blue copper proteins found in plants (Drew and Gatehouse, 1994;Guss et al, 1996;Hart et al, 1996;Nersissian et al, 1996;Vangysel et al, 1993). The former has been well established to participate in the photosynthetic electron transfer chain, connecting the cytochrome b, f complex and photosystem I (Hippler et al, 1996).…”

Copper Proteins with Type 1 SitesBased in part on the article Copper Proteins with Type 1 Sites by Edward N. Baker which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

“…The spectroscopic properties of cucumber stellacyanin are nearly identical to that of the protein from Rhus vernicifera. 132 …”