Fusion of a Coiled-Coil Domain Facilitates the High-Level Production of Catalytically Active Enzyme Inclusion Bodies

ChemCatChem ◽  
2015 ◽  
Vol 8 (1) ◽  
pp. 142-152 ◽  
Author(s):  
Martin Diener ◽  
Benita Kopka ◽  
Martina Pohl ◽  
Karl-Erich Jaeger ◽  
Ulrich Krauss
Keyword(s):  
Inclusion Bodies ◽  
Coiled Coil ◽  
Active Enzyme ◽  
Coiled Coil Domain ◽  
Catalytically Active ◽  
High Level ◽  
Level Production

High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies

Gene ◽  
1990 ◽  
Vol 87 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Yih-Shyun E. Cheng ◽  
Michell H. McGowan ◽  
Charles A. Kettner ◽  
John V. Schloss ◽  
Susan Erickson-Viitanen ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Active Enzyme ◽  
High Level Synthesis ◽  
High Level ◽  
Hiv 1

scholarly journals In Vivo Enzyme Immobilization by Inclusion Body Display

2010 ◽  
Vol 76 (16) ◽  
pp. 5563-5569 ◽  
Author(s):  
Björn Steinmann ◽  
Andreas Christmann ◽  
Tim Heiseler ◽  
Janine Fritz ◽  
Harald Kolmar
Keyword(s):  
Inclusion Body ◽  
Inclusion Bodies ◽  
Active Form ◽  
Coiled Coil ◽  
Cost Effective ◽  
Galactose Oxidase ◽  
Novel Strategy ◽  
High Level ◽  
Carboxy Terminal

ABSTRACT A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged α-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 μm, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.

Tailor-made catalytically active inclusion bodies for different applications in biocatalysis

2018 ◽  
Vol 8 (22) ◽  
pp. 5816-5826 ◽  
Author(s):  
Ramona Kloss ◽  
Tobias Karmainski ◽  
Vera D. Jäger ◽  
Doris Hahn ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Coiled Coil ◽  
Reaction Systems ◽  
Active Inclusion Bodies ◽  
Catalytically Active

CatIB properties can be tailored to the requirements of different reaction systems using two different coiled-coil domains as fusion tags.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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