Engineering fluorescent protein substrates for the AAA+ Lon protease

Wohlever, Matthew L.; Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.
April 2013
PEDS: Protein Engineering, Design & Selection;Apr2013, Vol. 26 Issue 4, p299
Academic Journal
AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal β-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.


Related Articles

  • Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing. Motejadded, Hassan; Altenbuchner, Josef // Biotechnology Letters;Apr2009, Vol. 31 Issue 4, p543 

    An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for...

  • Engineering and characterization of a superfolder green fluorescent protein. Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy; Terwilliger, Thomas C; Waldo, Geoffrey S // Nature Biotechnology;Jan2006, Vol. 24 Issue 1, p79 

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a...

  • Enzyme regulation: A thiol switch opens the gate. Antelmann, Haike // Nature Chemical Biology;Jan2015, Vol. 11 Issue 1, p4 

    The author discusses AAA+ proteases that are considered as quality control machineries consisting of substrate-binding adenosine triphosphatase (ATPase) modules for protein unfolding and a proteolytic chamber, and further reflects on a study that showed a redox switch in the Escherichia coli's...

  • New Approach to Achieve High-Level Secretory Expression of Heterologous Proteins by Using Tat Signal Peptide. Yu-Dong Li; Zhan Zhou; Long-Xian Lv; Xiao-Ping Hou; Yong-Quan Li // Protein & Peptide Letters;Jun2009, Vol. 16 Issue 6, p706 

    The twin-arginine translocation (Tat) pathway is an attractive route for secretory production of heterologous proteins in E. coli. In this study, we investigated the potential use of Tat signal peptide from S. coelicolor to improve secretory expression. The results showed that Tat signal peptide...

  • Regulatory Role of C-Terminal Residues of SulA in Its Degradation by Lon Protease in Escherichia coli1. Ishii, Yoshiyuki; Sonezaki, Shuji; Iwasaki, Yasushi; Miyata, Yukako; Akita, Kamml; Kato, Yasuhiko; Amano, Fumio // Journal of Biochemistry;2000, Vol. 127 Issue 5, p837 

    The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and...

  • Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry. Zhuoru Wu; Nieng Yan; Liang Feng; Oberstein, Adam; Hanchi Yan; Baker, Rosanna P.; Lichuan Gu; Jeffrey, Philip D.; Urban, Sinisa; Yigong Shi // Nature Structural & Molecular Biology;Dec2006, Vol. 13 Issue 12, p1084 

    Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine...

  • Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli. Ziyong Sun; Zhinan Xia; Feng Bi; Jian-Ning Liu // Applied Microbiology & Biotechnology;Mar2008, Vol. 78 Issue 3, p495 

    To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the...

  • Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Silverman, Joshua; Qiang Lu; Bakker, Alice; To, Wayne; Duguay, Amy; Alba, Ben M.; Smith, Richard; Rivas, Alberto; Li, Peng; Le, Hon; Whitehorn, Erik; Moore, Kevin W.; Swimmer, Candace; Perlroth, Victor; Vogt, Martin; Kolkman, Joost; Stemmer, Willem Pim C. // Nature Biotechnology;Dec2005, Vol. 23 Issue 12, p1556 

    We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating...

  • The surface protease OmpT serves as Escherichia coli Kl adhesion in binding to human brain micro vascular endothelial cells. Lei Wan; Yan Guo; Chang-Ye Hui; Xiao-Lu Liu; Wen-Bing Zhang; Hong Cao // Pakistan Journal of Pharmaceutical Sciences;May2014 Supplement, Vol. 27, p617 

    Escherichia coli (E. coli) Kl is the most conitnon bacteria that cause meningitis in the neonatal period. But it's not entirely clear ahout how E. coli crosses the blood-brain barrier. The features of the OmpT deletion in meningitic E. coli infection were texted in vitro. In comparison with the...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics