Expression of active hen egg white lysozyme in transgenic tobacco

Abstract

A full-length complementary DNA clone of hen egg white lysozyme (HEWL) was inserted into pBI121 vector lacking the β-glucuronidase (GUS) gene and used to transform tobacco. The HEWL gene had its own 18-amino acid signal peptide. HEWL activity was detected using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. The enzyme was active against Micrococcus luteus cells and migrated at the same molecular mass as purified mature HEWL. The protein was also found by immunodetection of HEWL after SDS-PAGE. Twenty-seven independent transgenic tobacco plants were analyzed for the expression of HEWL by a modified lysoplate assay for quantifying HEWL activity. Twenty-five plants exhibited HEWL activity up to 30 ng of HEWL per mg of leaf tissue. When using successive intercellular fluid (IF) extracts to study the localization of HEWL, it was found that no more than 10% of HEWL could be recovered in IF extracts. This is contrary to several extracellular proteins, such as the pathogenesis-related proteins. However, purified HEWL was recovered with the same yield as in transgenic tobacco leaves when it was injected into normal leaf tissue. Transgenic tobacco plants did not exhibit a phenotype change due to the expression of HEWL. The HEWL gene could be a useful reporter gene because the activity of the protein can be quantified by a simple assay.