A Modular Polycistronic Expression System for Overexpressing Protein Complexes in Escherichia coli

Department of Biochemistry and Molecular Biology, Center for Gene Regulation, University Park, Pennsylvania 16802-1014, USA.
Protein Expression and Purification (Impact Factor: 1.7). 03/2001; 21(1):224-34. DOI: 10.1006/prep.2000.1363
Source: PubMed


To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. I demonstrate successful use of the expression system for binary and ternary complexes, including the reconstitution of the VHL-elonginC-elonginB complex in E. coli and purification of the complex by affinity and ion-exchange chromatography. This polycistronic expression system should provide an important alternative to in vitro reconstitution of multicomponent complexes.

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    • "The polycistronic expression plasmid pST39/pET3aTr (a generous gift from Song Tan, Pennsylvania State University, University Park, PA) was used to produce the plasmids pST39- RBX1-HisCullin5, pST39-RBX2-HisCullin5 and pST39-CBF-beta- EloB-EloC-HisVif for co-expression of protein complexes in Escherichia coli. In these plasmids, the coding sequences of the following proteins were subcloned into the indicated restriction sites: RBX (RBX1/RBX2), XbaI and BamHI; HisCullin5, BspEI and MluI; CBF-b, XbaI and BamHI; EloB, EcoRI and HindIII; EloC, SacI and KpnI; and HisVif, BspEI and MluI [22]. VR-RBX1, VR-RBX2 and VR-Cullin5 were generated by subcloning RBX1, RBX2 and Cullin5, respectively, into the VR1012 vector at the PstI and BamHI restriction sites. "
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    ABSTRACT: Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Apr 2015 · Biochemical and Biophysical Research Communications
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    • "The glutathione resin was equilibrated in lysis buffer and proteins were eluted with 25 mM Tris- HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 5 mM glutathione. Rpt6-NT-MBP and Rpt3-NT-GST were coexpressed in E. coli with or without His6x-Adc17 using the pOPC/pOPT polycistronic plasmids (Fö rster et al., 2009; Ghislain et al., 1993; Tan, 2001; Tomko et al., 2010) and purified on amylose resin (New England Biolabs). Expression was performed as described above. "
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    • "In case of single-plasmid constructs, one can follow different strategies: One of these strategies includes the use of a single promoter in one plasmid, linking the two genes by a ribosome-binding site (RBS) similarly to genomic E. coli operons. This gene arrangement is also called bicistronic (two genes) or polycistronic (more than two genes) as termed in other studies [12] [13] [14]. This will yield one single transcript containing the information for the genes to be transcribed . "
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    ABSTRACT: L-asparaginases hydrolyze L-asparagine to L-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses L-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits α and β) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in E.coli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the β-subunit complexed to the α-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the β-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.
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