title: Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity.
abstract: Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).
authors: Department of Molecular Biosciences and Bioengineering, University of Hawai'i, Honolulu, HI, USA.
authors: Bei
authors: B
authors: Zhang
authors: Madhusudhan
authors: M
authors: Rapolu
authors: Leyang
authors: L
authors: Huang
authors: Wei Wen
authors: WW
authors: Su
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D059828:Plant Cells / Q000201:enzymology* / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D055571:Secretory Pathway; D020539:Sequence Analysis, Protein; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D017898:Tobamovirus / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014170:Transformation, Genetic; D019076:Transgenes; D000097573:Red Fluorescent Protein
chemical_list: D003470:Culture Media; D004792:Enzyme Precursors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020815:Polyproteins; D043484:Proprotein Convertases; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein; C058751:KEX2 protein, S cerevisiae