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Expression, Purification and Characterization of RecJ Nuclease -An Integrated Biochemistry Laboratory Course for Undergraduates

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  • Fengsong Cong

    (Department of Biochemistry and Molecular Biology, School of life Sciences and Biotechnology, Shanghai Jiaotong University, China)

Abstract

Here we describe a half-semester biochemistry laboratory course based on the expression, isolation, purification, and biochemical characterization of RecJ nuclease. The students transformed the expression plasmid of thermostable RecJ nuclease into the host bacteria Escherichia coli BL21(DE3), to induce the expression of the recombinant RecJ nuclease by IPTG. The expressed RecJ nuclease was purified from the cell lysates broken with sonication by Ni-affinity chromatography. SDS-denatured electrophoresis was utilized to identify the purity and size of the RecJ exonuclease. The activity of the enzyme was assessed by an in vitro exonuclease reaction using a fluorescent-labeled single-stranded DNA substrate. Through this lab course, students grasp the method of preparing and characterizing a recombinant protein and excise more than one biochemistry and molecular biology common techniques, which are great useful and significant to their future works.

Suggested Citation

  • Fengsong Cong, 2023. "Expression, Purification and Characterization of RecJ Nuclease -An Integrated Biochemistry Laboratory Course for Undergraduates," Biomedical Journal of Scientific & Technical Research, Biomedical Research Network+, LLC, vol. 50(3), pages 41743-41749, May.
    • Handle: RePEc:abf:journl:v:50:y:2023:i:3:p:41743-41749
    DOI: 10.26717/BJSTR.2023.50.007966
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