Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system

• Published in: PloS one Vol. 12; no. 3; p. e0174804
• Main Authors: Sun, He, Zhou, Ni, Wang, Hai, Huang, Dafang, Lang, Zhihong
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 30.03.2017; Public Library of Science (PLoS)
• Subjects: Algae; Amino acids; Analysis; Antifungal activity; Antimicrobial peptides; Bacteria; Barley; Biology and Life Sciences; Biotechnology; Breeding; C-Terminus; Capsid protein; Cell fusion; Cell recognition; Cleavage; Construction; Corn; Crop improvement; Crops; Diagnosis; E coli; Efficiency; Engineering; Enzymatic activity; Expression vectors; Fluorescence; Foot & mouth disease; Foot and mouth disease virus; Foot-and-Mouth Disease Virus - genetics; Foot-and-Mouth Disease Virus - metabolism; Fusion protein; Gene expression; Gene fusion; Genes; Genetic engineering; Genetic transformation; Genetically altered foods; Glucuronidase - genetics; Glucuronidase - metabolism; Green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Impatiens balsamina; Insects; Localization; Medical research; Mercury; Monoclonal antibodies; N-Terminus; Nutrition; Open reading frames; Open Reading Frames - genetics; Peptides; Peptides - genetics; Peptides - metabolism; Plant breeding; Plant layout; Plant tissues; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Plasmids; Polyproteins - genetics; Polyproteins - metabolism; Post-translation; Protein Processing, Post-Translational; Proteins; Protoplasts; Quantitation; Research and Analysis Methods; Rice; Sediment pollution; Seeds; Signal peptides; Soil contamination; Studies; Tobacco; Transgenic plants; Translation; Viral Proteins - genetics; Viral Proteins - metabolism; Viruses; Zea mays; Zea mays - genetics; Zea mays - metabolism; Beijing China; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0174804

In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF) incorporating two report genes, Green Fluorescent Protein (GFP) and β-glucuronidase (GUS), separated by 2A (or modified 2A), LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP) with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target localization in maize protoplast transformed by LP4/2A polyprotein construct and it implied the LP4/2A linker peptide could alleviate the inhibition of 2A processing by the carboxy-terminal region of upstream protein of 2A when translocated into the ER.