Cloning and expression of human defensin HNP-1 genomic DNA in Escherichia coli

Hiromu Takemura, Mitsuo Kaku, Shigeru Kohno, Hironori Tanaka, Ryoji Yoshida, Kazuo Ishida, Ryusuke Mizukane, Hironobu Koga, Kohei Hara, Toshiaki Usui, Takayuki Ezaki

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

We amplified a 181 bp DNA fragment encoding HNP-1 from genomic DNA of human polymorphonucleated neutrophils by PCR. Sequencing of this fragment revealed thai it only contained the mature protein coding region of HNP-1 and no intron was found in the sequences. The PCR product of HNP-1 genomic DNA was then cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (the GST-HNP-1 fusion protein). The GST-HNP-1 fusion protein was expressed as insoluble inclusion bodies, so they were collected and solubilized in 8 M urea. Thus, reasonably pure GST HNP-1 fusion protein was obtained, with which rabbits were immunized to raise polyclonal antibody against HNP-1. Ouchterlony immunodiffusion and immunoblotting showed that the antiserum reacted with chemically synthesized HNP-1, -2 and human PMN extracts and did not cross react with human GST in these assays. We demonstrated that human defensin HNP-1, a small cationic protein, was expressed in E. coli as an insoluble GST fusion protein, and polyclonal antibody raised by immunizing rabbits with this fusion protein did not cross react with human GST and was specific to defensins.

Original languageEnglish
Pages (from-to)287-293
Number of pages7
JournalJournal of Microbiological Methods
Volume25
Issue number3
DOIs
Publication statusPublished - 1996 Jun

Keywords

  • Cloning
  • Defensin
  • Escherichia coli
  • Expression
  • HNP-1 gene
  • pGEX-2T

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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