[Prokaryotic expression and identification of dual-fluorescence fusion proteins of small ubiquitin-like modifier and sentrin-specific protease].
We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Wang
Xiaohui
X
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. xnwang@21cn.net
Guo
Jie
J
Wang
Jufang
J
Li
Shan
S
Sun
Lihua
L
Wang
Xiaoning
X
Lü
Jianxin
J
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D025842:SUMO-1 Protein / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases; C501833:SENP2 protein, human