Novel Autoprocessing Domains for Stoichiometric Protein Co-Expression in Plants

Abstract

Coordinate expression of multiple proteins in plants plays important roles in both fundamental research and commercial practice in plant biotechnology. However, current technologies amenable to precise, efficient and stoichiometric multi-protein expressions are not yet available. This study aims to advance current state-of-the-art in multi-protein co-expression by developing novel polyprotein-based expression vector systems. These vectors enable stoichiometric expression of multiple proteins from a single open reading frame (ORF) that encodes a polyprotein precursor which is composed of multiple proteins connected by an autoprocessing domain. The unique autoprocessing domains allow individual proteins to be separated from the polyprotein precursor via either co-translational or post-translational cleavage by endogenous host proteases or self-catalytic cleavage activity. As a result, nearly stoichiometric expression of multiple proteins can be achieved using a single expression cassette. In this study, three independent polyprotein cassettes mediated by respective autoprocessing domains: (1) kex2p-like protease substrate sequence, (2) dual-intein and (3) intein-FMDV2A domain, were devised and investigated for coordinate co-expression of multiple proteins in plants. Efficient release of individual protein moiety was demonstrated in tobacco cells as well as in different plant species using immunoblotting analysis. Cellular cleavage mechanisms of the polyprotein precursors were elucidated by N-terminal amino acid sequencing along with mass spectrometry analysis. Functionality of released proteins was characterized by fluorescence spectroscopy and bioactivity assays. Specific cellular targeting of released proteins can be achieved by incorporation of suitable signal sequences. Collectively, our results have successfully demonstrated the utility of different engineered autoprocessing domains for efficient co-expression of multiple proteins from novel polyprotein vectors. This technology should be highly useful in a wide range of applications, from improving fundamental studies of molecular mechanisms, to enhance crop yield, stress/disease tolerance, nutritional values, and production of high-value proteins.