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Updated: Jul 18, 2026
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
[Efficient expression and purification of human beta-defensin-2 in E.coli]
Zhi-xia Zhong1, Hong Ruan, Li-mei Fan
1Institute of Bioengineering, Zhejiang University, Hangzhou 310027, China.
Objective:
To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2.
Methods:
DNA fragment containing mature hBD2 coding region (smhBD2-cDNA) was amplified by PCR, multiple copies of smhBD2-cDNA were linked using Bgl II and BamH I enzymes, pET32-nsmHBD2-cDNA with 1, 2, 4, or 8 copies of smhBD2-cDNA was constructed. The soluble and insoluble hBD2 proteins were separated and analyzed by SDS-PAGE analysis. The soluble protein underwent a separation process containing affinity chromatography, enterokinase digestion and ion exchange chromatography to get the recombinant hBD2 peptide. The bioactivity of recombinant hBD2 was examined by bacteria-inhibition tests in liquid culture.
Result:
The plasmids pET32-nsmHBD2-cDNA with 1, 2, 4 copies of smhBD2-cDNA were constructed and the expressed soluble protein accounted for 52 %, 48 %, and 31 % respectively. The plasmids with 8 copies expressed mainly insoluble protein with few in soluble form. The growth of E.coli K12D31 was dramatically suppressed with a inhibition rate of 90 %, when the final concentration of recombinant hBD2 reached between 0.4 to 0.5 mug/ml.
Conclusion:
Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.
