title: A dual-intein autoprocessing domain that directs synchronized protein co-expression in both prokaryotes and eukaryotes.
abstract: Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine.
authors.affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Bei
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Madhusudhan
authors.initials: M
authors.lastname: Rapolu
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Liang
authors.affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Han
authors.affiliation: Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Williams
authors.affiliation: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Wei Wen
authors.initials: WW
authors.lastname: Su
mesh_terms: D056890:Eukaryota / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047668:Inteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D059828:Plant Cells / Q000378:metabolism; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D011387:Prokaryotic Cells / Q000378:metabolism*; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020815:Polyproteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein