Expression of a hybrid (1–3, 1–4)-β-glucanase in barley protoplasts

Abstract

Barley aleurone protoplasts were used to study expression and secretion of a heat-stable hybrid, Bacillus (1–3, 1–4)-β-glucanase (H(A107-M)). The strength and hormone responsiveness of a barley, aleurone specific, low p I α-amylase promoter was analysed in comparison with the synthetic, constitutive pEmu promoter, by expressing the reporter gene encoding chloramphenicol acetyl transferase (CAT) in aleurone protoplasts. Both promoters directed high levels of CAT expression after GA 3 addition. Plasmids containing either promoter upstream of the coding region for H(A107-M) were also constructed. To effect secretion of H(A107-M) the coding region for a low p I α-amylase signal peptide was inserted between each promoter and the (1–3, 1–4)-β-glucanase coding region. Activity derived from heat-stable (1–3, 1–4)-β-glucanase could not be detected in protoplasts transfected with the α-amylase promoter containing plasmids, whereas both plasmids with the pEmu promoter directed expression of H(A107-M). Measurements of (1–3, 1–4)-β-glucanase activity from protoplasts transfected with plasmids encoding the mature enzyme indicated that the majority of H(A107-M) was intracellular. When the plasmid encoding the pre-enzyme was used, 90% of the activity from H(A107-M) was extracellular. The intracellular H(A107-M) also reacted with specific antibodies, was active and had the expected molecular mass of 24 kDa. Extracellular H9A107-M) was post-translationally modified resulting in three active forms of 22, 24 and 28 kDa.