Construction of Mutants Swapping Ubiquitin E3 Ligase CHIP and E4B U-box Domain and Verification of Ubiquitination Activity

doi:10.13560/j.cnki.biotech.bull.1985.2021-0230

Abstract

Abstract:

This work aims to construct mutants of ubiquitin E3 ligases CHIP and E4B that exchange U-box domains and to study the effect of U-box domain on the activities of 2 U-box family ubiquitin ligases CHIP and E4B. By employing overlap PCR,a mutant CHIP named C+E that contained the U-box domain derived from E4B,and a mutant E4B named E+C whose U-box was replaced by the U-box domain from CHIP,were constructed respectively. These mutants’ plasmids and the substrate of CHIP named RCC2,the substrate of E4B named NIPSNAP1,and their common substrates p53 and CDC37 respectively were co-transfected into HEK-293T cells. Western Blot were adapted to detect the expressions of these substrates,CHIP,E4B and their mutants. Immunoprecipitation was to detect the ubiquitination of the substrates. The results showed that two mutants swapping the U-box domain were successfully constructed,and both of them were expressed in HEK-293T cells. Both mutants partially retained the activity on the ubiquitination of their respective substrates and C+E could have common substrates p53 and CDC37 in the ubiquitination.

Key words: ubiquitin E3 ligase CHIP, ubiquitin E3 ligase E4B, mutant, U-box domain, substrate

FAN Yu-chen, LU Yao, LIU Xiang-nan, ZHAO Bo. Construction of Mutants Swapping Ubiquitin E3 Ligase CHIP and E4B U-box Domain and Verification of Ubiquitination Activity[J]. Biotechnology Bulletin, 2021, 37(12): 191-197.

Figures/Tables 4

Fig.1 Plasmid cloning and construction of substrate RCC2 and p53 M:DNA molecular weight standard. A:PCR products of RCC2 gene. B:PCR products of p53 gene. C:Electrophoresis band of pLVX empty vector plasmid and positive cloned plasmid

Fig.2 PCR results of fusion genes C+E and E+C M:DNA molecular weight standard. A:PCR products of CA,CB,EA and EB fragments. B:PCR products of C+E gene. C:PCR products of E+C gene

Fig.3 Intracellular expression results of substrates,CHIP,E4B and their mutants M:Protein molecular weight standard. A,B,C and D:Expression levels of substrates,CHIP,E4B and their mutants in cell lysates

Fig.4 Co-immunoprecipitation experiment results of sub-strate RCC2,NIPSNAP1,p53 and CDC37 A,B,C and D:Detection of the ubiquitination activity of CHIP,E4B and their mutants on the substrates in the cell by co-immunoprecipitation

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