Cloning and Prokaryotic Expression of SoGSNOR from Spinach

doi:10.7668/hbnxb.2018.01.013

Abstract

Abstract: S-nitrosoglutathione reductase (GSNOR) can modulate metabolism and homeostasis of NO in organisms. In order to further study GSNOR function, the full-length cDNA encoding SoGSNOR was amplified by RT-PCR and RACE (Rapid-amplification of cDNA ends).The open reading frame of SoGSNOR was 1 140 bp,encoding 379 amino acids (GenBank accession No.KR381778).The SoGSNOR was inserted into pET32a.The correct recombinant pET32a-SoGSNOR was transformed into E.coli BL21 star (DE3).The SoGSNOR-His fusion protein was induced by isopropyl-beta-D-thiogalactoside (IPTG).SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 65 ku was highly expressed in E.coli BL21 star(DE3) and existed both in the supernatant and the precipitation part of E.coli lysates.The supernatant was further purified by Ni²⁺ NTA affinity chromatography and immunized white mouse as an antigen.The polyclonal antibody was obtained and analyzed by Western Blot and a specific band was detected which was corresponding to the expected size of SoGSNOR.The study laid a foundation for further investigating the function of SoGSNOR.

Key words: S-nitrosoglutathione reductase, Prokaryotic expression, Spinach

CLC Number:

Q78
S636.03

GUO Zhaolai, BAI Xuegui, LI Kunzhi, XU Huini. Cloning and Prokaryotic Expression of SoGSNOR from Spinach[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(1): 80-85. doi: 10.7668/hbnxb.2018.01.013.

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