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 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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    ABSTRACT: The proteasome is essential for the selective degradation of most cellular proteins. To survive overwhelming demands on the proteasome arising during environmental stresses, cells increase proteasome abundance. Proteasome assembly is known to be complex. How stressed cells overcome this vital challenge is unknown. In an unbiased suppressor screen aimed at rescuing the defects of a yeast Rpt6 thermosensitive proteasome mutant, we identified a protein, hereafter named Adc17, as it functions as an ATPase dedicated chaperone. Adc17 interacts with the amino terminus of Rpt6 to assist formation of the Rpt6-Rpt3 ATPase pair, an early step in proteasome assembly. Adc17 is important for cell fitness, and its absence aggravates proteasome defects. The abundance of Adc17 increases upon proteasome stresses, and its function is crucial to maintain homeostatic proteasome levels. Thus, cells have mechanisms to adjust proteasome assembly when demands increase, and Adc17 is a critical effector of this process.
    Molecular Cell 08/2014; 55(4). DOI:10.1016/j.molcel.2014.06.017 · 14.02 Impact Factor
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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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    Protein Expression and Purification 10/2013; 93. DOI:10.1016/j.pep.2013.10.007 · 1.70 Impact Factor
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    • "The polycistronic expression system pST39/pET3aTr (a generous gift from Song Tan, Pennsylvania State University, University Park, PA) was used to produce the plasmids pST39-Vif, pST39-EloB, pST39-EloC, pST39- CBF-β, pST39-Vif-CBF-β, pST39-Vif-EloB, pST39-Vif-EloC, pST39-Vif-EloB-EloC, pST39-Vif-EloB-CBF-β, pST39-Vif-EloC-CBF-β and pST39-Vif-EloB-EloC-CBF-β for expression of proteins or protein complexes in E. coli. In these plasmids, sequences encoding the following proteins were cloned between the indicated restriction sites: Vif, XbaI and BamHI; CBF-β, EcoRI and HindIII; EloC, SacI and KpnI; and EloB, BspEI and MluI [63]. The CBF-β gene was acquired by RT-PCR as described below. "
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    Retrovirology 08/2013; 10(1):94. DOI:10.1186/1742-4690-10-94 · 4.19 Impact Factor
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