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Updated: May 25, 2026
Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli
Published on: June 9, 2014
[Preparation and identification of recombinant human neutrophil elastase]
Jin-Chun Lu1, Kun-Gang Lu, Hong-Ye Zhang
1Department of Laboratory Science, Nanjing Hospital, Jiangsu Corps of the Armed Police Force, PLA, Nanjing, Jiangsu 210028, China. lujc@androl.cn
Objective:
To prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.
Methods:
HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.
Results:
The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.
Conclusion:
The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.
