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On-column Refolding of an Insoluble His6-tagged recombinant ec-sod Overexpressed in Escherichia coli

Acta Biochimica et Biophysica Sinica 2005年第4期37卷 265-269页

作者： Xi-Qiang ZHU Su-Xia LI Hua-Jun HE Qin-Sheng YUAN State Key Laboratory of Bioreactor Engineering and Institute of Biochemistry,East China University of Science and Technology,Shanghai 200237,China StateKeyLaboratoryofBioreactorEngineeringandInstituteofBiochemistry EastChinaUniversityofScienceandTechnologyShanghai200237

The ec-sod cDNA was cloned by polymerase chain reaction （PCR） and inserted into theEscherichia coli expression plasmid pET-28a（+） and transformed into *** BL21 （DE3）.The correspondingprotein that was overexpressed as a recombinant His6-tagged ec-sod was present in the form of inactiveinclusion *** structure was first solubilized under denaturant conditions （8.0 M urea）.Then,after acapture step using immobilized metal affinity chromatography （IMAC）,a gradual refolding of the protein wasperformed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione（GSH）and oxidized glutathione（GSSG）.The mass ratio of GSH to GSSG was 4:*** purified enzyme was active,showing that at least part of the protein was properly *** protein was made concentrated byultrafiltration,and then isolated using Sephacryl S-200 *** were two protein peaks in the A280 *** on the results of electrophoresis,we concluded that the two fractions were formed by protein subunitsof the same mass,and in the fraction where the molecular weight was higher,the dimer was formed throughthe disulfide bond between *** were detected in the two fractions,but the activity of thedimer was much higher than that of the single *** special activities of the two fractions werefound to be 3475 U/mg protein and 510 U/mg protein,respectively.

关键词： refolding His6 tag recombinant ec-sod inclusion body protein subunit structure

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