Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain

PIST [PDZ (PSD-95, Discs-large and ZO-1) protein interacting specifically with TC10] functions as a regulator of membrane trafficking with Rab6A. Recently, the involvement of the fusion of PIST with ROS1 in cancer development has been identified. In this study, the coiled-coil domain of PIST, which is the domain responsible for interaction with Rab6A and fusion with ROS1, corresponding to amino acids 29–133, was overexpressed inEscherichia coliusing engineered C-terminal His tags. The coiled-coil domain of PIST was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 4.0 Å from a crystal belonging to the hexagonal space groupP6222 orP6422, with unit-cell parametersa=b= 85.19,c= 240.09 Å, γ = 120.00°.

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

Biochemical Journal ◽

10.1042/bcj20190359 ◽

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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Faculty Opinions recommendation of The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717967809.793467391 ◽

2012 ◽

Author(s):

John Tainer

Keyword(s):

Coiled Coil ◽

Coiled Coil Domain ◽

Mre11 Complex ◽

Complex Functions

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The git5 Gβ and git11 Gγ Form an Atypical Gβγ Dimer Acting in the Fission Yeast Glucose/cAMP Pathway

Genetics ◽

10.1093/genetics/157.3.1159 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1159-1168 ◽

Cited By ~ 5

Author(s):

Sheila Landry ◽

Charles S Hoffman

Keyword(s):

Fission Yeast ◽

Mammalian Cells ◽

Glucose Repression ◽

Coiled Coil ◽

Carboxy Terminus ◽

Amino Terminal ◽

Camp Pathway ◽

Coiled Coil Domain ◽

Mutational Activation ◽

Two Hybrid

AbstractFission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Gα and git5 Gβ are both required for glucose-triggered cAMP signaling. The git5 Gβ is a unique member of the Gβ family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gβ folding and interaction with Gγ subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Gγ gene. Co-immunoprecipitation studies confirm the composition of this Gβγ dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Gα or the git5 Gβ. Overexpression of the gpa2 Gα partially suppresses loss of either the git5 Gβ or the git11 Gγ, while mutational activation of the Gα fully suppresses loss of either Gβ or Gγ. Deletion of gpa2 (Gα), git5 (Gβ), or git11 (Gγ) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Gα subunit remains partially active in the absence of the Gβγ dimer and that the git5 Gβ subunit remains partially active in the absence of the git11 Gγ subunit. The addition of the CAAX box from the git11 Gγ to the carboxy-terminus of the git5 Gβ partially suppresses the loss of the Gγ. Thus the Gγ in this system is presumably required for localization of the Gβγ dimer but not for folding of the Gβ subunit. In mammalian cells, the essential roles of the Gβ amino-terminal coiled-coil domains and Gγ partners in Gβ folding may therefore reflect a mechanism used by cells that express multiple forms of both Gβ and Gγ subunits to regulate the composition and activity of its G proteins.

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Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design

Catalysts ◽

10.3390/catal11020176 ◽

2021 ◽

Vol 11 (2) ◽

pp. 176

Author(s):

Sophie Vincenti ◽

Magali Mariani ◽

Jessica Croce ◽

Eva Faillace ◽

Virginie Brunini-Bronzini de Caraffa ◽

...

Keyword(s):

Escherichia Coli ◽

Expression System ◽

Scale Up ◽

Food Additives ◽

Statistical Design ◽

Green Leaf Volatiles ◽

Hydroperoxide Lyase ◽

Soluble Enzyme ◽

Leaf Volatiles ◽

Conversion Rates

Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their “fresh green” aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box–Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L−1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L−1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported.

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