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
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.
Available from: Anne Bertolotti
- "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. "
[Show abstract] [Hide abstract]
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
Available from: Christos S Karamitros
- "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   . This will yield one single transcript containing the information for the genes to be transcribed . "
[Show abstract] [Hide abstract]
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.
Protein Expression and Purification 10/2013; 93. DOI:10.1016/j.pep.2013.10.007 · 1.70 Impact Factor
Available from: Mingyu Lv
- "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 . The CBF-β gene was acquired by RT-PCR as described below. "
[Show abstract] [Hide abstract]
The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex.
The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-β binding. (3) EloB-EloC but not CBF-β could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14.
Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization.
Retrovirology 08/2013; 10(1):94. DOI:10.1186/1742-4690-10-94 · 4.19 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.