Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites

Chen, Wen-Shuo; Chang, Y. C.; Chen, Ying‐Ju; Chen, Yujie; Teng, Chao-Yi; Wang, Chung-Hsiung; Wu, Tzong‐Yuan

doi:10.1016/j.jviromet.2009.03.019

Cited by 12 publications

(5 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Coexpression toolbox for production of protein complexes Expression systems for production of protein complexes in E. coli frequently make use of polycistronic expression cassettes with several genes of interest, spaced apart by ribosome binding sites (Shine-Dalgarno sequences), placed under the control of a single promoter, and typically followed by a sequence for efficient termination of mRNA transcription [10,11,21,[60][61][62] (Figure Ia). In eukaryotic hosts, an analogous design can involve internal ribosomal entry sites (IRESs), which are inserted between gene coding regions under control of a single promoter [24][25][26][27] ( Figure Ib). IRESs exist in the 5 0untranslated regions of many viral and cellular mRNAs [24], and facilitate cap-independent translation by recruiting ribosomes for efficient protein production.…”

Section: Multiprotein Complexes Catalyze Cellular Functionmentioning

confidence: 99%

“…IRES sequences differ greatly and exhibit species specificity [26]. For example, IRES elements from encephalomyocarditis virus (EMCV) work well in mammalian cells, whereas IRESs from Perina nuda virus (PhV) and Rhopalosiphum padi virus (RhPV) have been successfully used for protein complex production in insect cells [27].…”

Section: Multiprotein Complexes Catalyze Cellular Functionmentioning

confidence: 99%

“…In addition to MultiBac, a growing number of technologies are currently being used for expressing protein complexes (Box 1). Examples of these approaches are the use of internal ribosome entry sites (IRESs) for multigene expression, and the use of polyprotein constructions involving self-cleaving peptide sequences or proteolytic processing of large precursor polypeptides by specific proteases [22,[24][25][26][27][28][29][30]. Particularly promising for structural biology, where often a large number of constructs need to be scrutinized in parallel, are new methods coupling gene libraries to coexpression (Box 1) [31].…”

Section: Reviewmentioning

confidence: 99%

See 2 more Smart Citations

MultiBac: expanding the research toolbox for multiprotein complexes

Bieniossek

Imasaki

Takagi

et al. 2012

Trends in Biochemical Sciences

213

227

View full text Add to dashboard Cite

Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.

show abstract

Section: Multiprotein Complexes Catalyze Cellular Functionmentioning

confidence: 99%

Section: Multiprotein Complexes Catalyze Cellular Functionmentioning

confidence: 99%

Section: Reviewmentioning

confidence: 99%

See 1 more Smart Citation

MultiBac: expanding the research toolbox for multiprotein complexes

Bieniossek

Imasaki

Takagi

et al. 2012

Trends in Biochemical Sciences

213

227

View full text Add to dashboard Cite

show abstract

“…Multiple vectors strategy led to inefficient expression and difficulty of transfection, while multiple promoters or polycistron strategy may result in suppressed down-stream transcription or undesirable expression proportions (Villemure et al, 2001;Dzivenu et al, 2004;Patial et al, 2007;Chen et al, 2009). We overcame this impediment since multiple components came from a single long transcript and accumulated to high level with appropriate proportions in our system.…”

Section: Discussionmentioning

confidence: 99%

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

et al. 2012

View full text Add to dashboard Cite

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His6 tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

show abstract

“…Unfortunately, this method is susceptible to gene suppression as a result of promoter interference 4. The internal ribosomal entry site mediated polycistronic vector is an improvement to the aforementioned multiple expression vector as it expresses all its genes from a single open reading frame through a translation reinitiation mechanism 5. However, the translations are uncoupled in the sense that they are invoked from different translational initiation events.…”

Section: Introductionmentioning

confidence: 99%

TEV protease‐facilitated stoichiometric delivery of multiple genes using a single expression vector

2010

View full text Add to dashboard Cite

Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self-cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS-PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self-processing vector.

show abstract

Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites

Cited by 12 publications

References 37 publications

MultiBac: expanding the research toolbox for multiprotein complexes

MultiBac: expanding the research toolbox for multiprotein complexes

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

TEV protease‐facilitated stoichiometric delivery of multiple genes using a single expression vector

Contact Info

Product

Resources

About