Plasmid Construction for Bacterial and Mammalian Expression

We generated plasmids encoding various truncated mutants of UCP, pVHL and wild-type UbcH5c for expression in bacterial cells or mammalian cells [9 (link)]. For bacterial expression, plasmids were constructed by ligating PCR products into pET28a (Novagen, WI, USA) and pGEX-4T-1 (GE Healthcare Life Sciences, WI, USA), and for mammalian expression, pFlag-CMV1 (Sigma-Aldrich, MO, USA), pEBG and pcDNA3-HA (Invitrogen, CA, USA) were used. Mutants of UCP and VHL were generated based on the wild-type genes using a PCR method [16 (link)].

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Lim J.H., Shin H.W., Chung K.S., Kim N.S., Kim J.H., Jung H.R., Im D.S, & Jung C.R. (2016). E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue. PLoS ONE, 11(9), e0163710.

Publication 2016

pGEX-4T-1 pFlag-CMV1 pcDNA3-HA

Bacterial Cells Genes Mammalian Plasmids

Corresponding Organization : Korea University of Science and Technology

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Variable analysis

independent variables

Various truncated mutants of UCP
Various truncated mutants of pVHL
Wild-type UbcH5c

dependent variables

Expression of UCP, pVHL, and UbcH5c in bacterial cells or mammalian cells

control variables

Plasmid vectors used for bacterial expression (pET28a and pGEX-4T-1)
Plasmid vectors used for mammalian expression (pFlag-CMV1, pEBG, and pcDNA3-HA)

controls

Wild-type genes of UCP and VHL used as controls for generating mutants

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