Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

Hondred, David; Walker, Joseph; Mathews, Dennis E.; Vierstra, Richard D.

doi:10.1104/pp.119.2.713

Cited by 77 publications

(70 citation statements)

References 55 publications

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“…Similarly, UBC 8-11 exhibit nearly identical abilities to stimulate substrate-independent ubiquitination of a variety of RING type E3s in vitro (Kraft et al, 2005). UBC8 is able to catalyze ubiquitination with a HECT E3, a distinct E3 type (see below), while UBC1, UBC4 and UBC7, members of other subfamilies, were not (Bates and Vierstra, 1999). For these reasons, members of this E2 subfamily are often referred to as 'generic' E2s and are the E2s that should be utilized in tests for E3 activity in the absence of any other information.…”

Section: Ubiquitin Carrier Proteins (Ubiquitin Conjugating Enzymes Umentioning

confidence: 99%

“…Separately, it was noted that co-synthesis with ubiquitin in E. coli enhanced a protein's solubility, suggesting this method of synthesis as a tool to enhance protein yield in E. coli in general (Varshavsky, 2005). In addition, ubiquitin fusions have been proposed to enhance protein expression in Arabidopsis (Hondred et al, 1999) and as a method to produce multiple proteins from the same mRNA (Walker and Vierstra, 2007).…”

Section: Ubiquitin Genesmentioning

confidence: 99%

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The Ubiquitination Machinery of the Ubiquitin System

Callis

2014

The Arabidopsis Book

293

269

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The protein ubiquitin is a covalent modifier of proteins, including itself. The ubiquitin system encompasses the enzymes required for catalysing attachment of ubiquitin to substrates as well as proteins that bind to ubiquitinated proteins leading them to their final fate. Also included are activities that remove ubiquitin independent of, or in concert with, proteolysis of the substrate, either by the proteasome or proteases in the vacuole. In addition to ubiquitin encoded by a family of fusion proteins, there are proteins with ubiquitin-like domains, likely forming ubiquitin's β-grasp fold, but incapable of covalent modification. However, they serve as protein-protein interaction platforms within the ubiquitin system. Multi-gene families encode all of these types of activities. Within the ubiquitination machinery "half" of the ubiquitin system are redundant, partially redundant, and unique components affecting diverse developmental and environmental responses in plants. Notably, multiple aspects of biotic and abiotic stress responses require, or are modulated by, ubiquitination. Finally, aspects of the ubiquitin system have broad utility: as components to enhance gene expression or to regulate protein abundance. This review focuses on the ubiquitination machinery: ubiquitin, unique aspects about the synthesis of ubiquitin and organization of its gene family, ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases, or E3s. Given the large number of E3s in Arabidopsis this review covers the U box, HECT and RING type E3s, with the exception of the cullin-based E3s.

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Section: Ubiquitin Carrier Proteins (Ubiquitin Conjugating Enzymes Umentioning

confidence: 99%

Section: Ubiquitin Genesmentioning

confidence: 99%

The Ubiquitination Machinery of the Ubiquitin System

Callis

2014

The Arabidopsis Book

293

269

View full text Add to dashboard Cite

show abstract

“…Amplification of the PAE2 mRNA was used as a control (Downes et al, 2003). RNA gel blot analysis was performed with total RNA isolated by aurintricarboxylic acid/phenol/chloroform extraction followed by sequential lithium chloride and ethanol/potassium acetate precipitations (Hondred et al, 1999). 32 P-labeled RNA probes were synthesized by the riboprobe system using Sp6 or T7 RNA polymerase (Promega) and the EBF1, EBF2, EIN3, and EIL1 cDNAs as templates.…”

Section: Rna Isolation and Analysismentioning

confidence: 99%

The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling

et al. 2007

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Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1-based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.

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“…The use of Ub to enhance the levels of protein expression has been reported previously [4,13,14,[40][41][42][43]. In the present study, efficient expression of the Ub::hGH fusion gene in E. coli strains was achieved, and the UBPD2C analog of the UBP1 protease was used to cleave the Ub moiety from the N-terminus of the hGH.…”

Section: Resultsmentioning

confidence: 67%

Use of Ubp1 protease analog to produce recombinant human growth hormone in

et al. 2014

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Background: Numerous bacterial human growth hormone (hGH) expression methods under conventional fermentation and induction conditions have been described. Despite significant progress made in this area over the past several years, production of recombinant hGH by using cellular expression systems still requires further optimization. Fusion of the ubiquitin (Ub) tag to the hGH protein allowed to increase of the overall efficiency of the biosynthesis and improve the protein stability. Ub is a protein composed of 76 amino acid residues with a molecular mass of 8.6 kDa, expressed in all eukaryotes. This protein is an element of the universal protein modification system, which does not occur in bacteria, and is a useful carrier for heterologous proteins obtained through expression in Escherichia coli. Purification of Ub-fusion proteins is easier than that of unconjugated recombinant proteins, and Ub can be removed by deubiquitinating proteases (DUBs or UBPs).

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Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

Cited by 77 publications

References 55 publications

The Ubiquitination Machinery of the Ubiquitin System

The Ubiquitination Machinery of the Ubiquitin System

The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling

Use of Ubp1 protease analog to produce recombinant human growth hormone in

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