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.
Highlights
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
Three strategies are most commonly practiced for co-expression of multiple proteins: (i) multiple monocistronic expression cassettes on the same or separate vectors; (ii) polycistronic vectors based on ribosome binding site (RBS) or internal ribosomal entry site (IRES) for prokaryotic and eukaryotic hosts, respectively; and (iii) polyprotein vectors based on foot-and-mouth-disease-virus (FMDV) 2A-like peptide or protease substrate sequence for eukaryotic hosts[12,13]
When linked transgene expression cassettes www.nature.com/scientificreports are used on a single vector, because each gene cassette harbors its own set of regulatory elements, the size of the vector could become quite large especially if the transgene number increases
Summary
Bei Zhang[1], Madhusudhan Rapolu[1], Zhibin Liang[2], Zhenlin Han[1], Philip G. 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. Intein is a naturally found protein splicing element that undergoes self-excision from a protein precursor with concomitant joining (splicing) of the flanking protein sequences called exteins This protein splicing process is autocatalytic and does not require the presence of any exogenous host-specific proteases or co-factors. We report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dualintein (DI) domain, to achieve coordinated co-expression of multiple proteins. We investigated the DI system in bacterial, mammalian, and plant hosts, and confirmed its usefulness in all host systems tested
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