Author: Kafeel Ahmad, David Twell, Kevin Christopher Gough and Ben Charles Maddison
Publishing Date: 2017
Volume 30 Issue 2
In order to express multisubunit proteins, or to manipulate metabolic pathways in plants it is essential to be able to efficiently express multiple proteins within the same plant cell. To increase the efficiency of multi-protein expression, we demonstrate the use of the Golgi localized Kex2 protease activity in tobacco to process a large polyprotein precursor consisting of four individual protein domains into its individual protein constituents. Four genes encoding enzymes involved in the biosynthesis of S. pneumoniae type 2 polysaccharide were assembled into a single expression cassette as a large polyprotein driven by a single cauliflower mosaic virus (CaMV) 35S promoter. Each of the individual protein domains were separated by three sequential Kex2 protease digestion sites. At the N-terminus a Pr1b signal peptide was incorporated for efficient targeting of the polyprotein to the apoplast. Each individual protein domain was tagged with its own immuno-tag. The construct was used for the transformation of Nicotiana tabacum and stable lines were selected. All four processed proteins could be immunologically detected in protein extracts using Western blotting indicating correct expression and Kex2 processing. Utilisation of the Kex2 protease system represents an efficient way of expressing multiple proteins in the same plant. This method simplifies the transformation procedures, and presents a method for expression of multiple proteins within the same plant.
KEYWORDS: Kex2 protease, S. pneumoniae, polyprotein precursor, transgenic