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Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli

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Abstract

LECT2 is a chemotactic protein to neutrophils. A recombinant six-histidine-tagged human LECT2, (His)6-LECT2, was expressed in E. coli using a pET21a(+) vector. The (His)6-LECT2 was purified from the soluble fraction in E. coli as a single band in sodium dodesyl sulfate/polyacrylamide gel electrophoresis using three steps of column chromatography with Ni2+-charged nitrilo-triacetic acid (Ni-NTA) agarose, DEAE-Sepharose, and CM-Sepharose. The purified (His)6-LECT2 was yielded with 96 µg from the soluble fraction of 1,500 ml culture of E. coli. The circular dichroism spectrum of (His)6-LECT2 showed the folded structure, which is rich in β-sheet structure and rare in α-helix.

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References

  • Crowe J, Dobeli H, Gentz E, Hochuli, Stuber D and Henco K (1994) 6 × His-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol. Biol. 31: 371–387.

    PubMed  CAS  Google Scholar 

  • Foutolakis M, Kania M, Ozmen L, Loetscher HR, Garotta G and van Loon AP (1989) Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IBF-gamma. One single 90-kilodalton INF-gamma receptor can lead to multiple cross-linked products and isolated proteins. J. Immunol. 143: 3266–3276.

    Google Scholar 

  • Gray MR, Colot HV, Guarente L and Rosbash M (1982) Open reading frame cloning: Identification, cloning, and expression of open reading frame DNA. Proc. Natl. Acad. Sci. USA, 79: 6598–6602.

    Article  PubMed  CAS  Google Scholar 

  • Hochuli E, Dobeli H and Schacher A (1987) New metal chelate absorbant selective for proteins and peptides containing neighbouring histidine residues. J. Chromato. 411: 177–184.

    Article  CAS  Google Scholar 

  • Kaslow DC and Shiloach J (1994) Production, purification and immunogenicity of a malaria transmission-blocking vaccine candidate: TBV25H expressed in yeast and purified using nickel-NTA agarose. Biol. Technol. 12: 494–497.

    CAS  Google Scholar 

  • Koshio O, Mizuno S and Suzuki K (1990) Formyl-Met-Leu-Phe-dependent serine kinase for a 64000 molecular weight protein of polymorphonuclear leukocytes in a cell-lysate system. Cell. Signaling 2: 471–477.

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  PubMed  CAS  Google Scholar 

  • Maina CV, Riggs PD, Grandea AG III, Slatko BE, Laurie S, Moran LS, Tagliamonte JA, McReynols LA and di Guan C (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74: 365–373.

    Article  PubMed  CAS  Google Scholar 

  • Parks TD, Leuther KK, Howard ED, Johnston SA and Dougherty WF (1994) Release of proteins and peptides from fusion proteins using a recombinant plant virus protainase. Anal. Biochem. 216: 413–417.

    Article  PubMed  CAS  Google Scholar 

  • Provencher SW and Glockner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20(1): 33–37.

    Article  PubMed  CAS  Google Scholar 

  • Suzuki K, Miyasaka H, Ota H, Yamakawa Y, Tagawa M, Kuramoto A and Mizuno S (1989) Purification and partial sequence of a chemotactic protein for polymorphonuclear leukocytes derived from human lung giant cell carcinoma LU65C cells. J. Exp. Med. 169: 1085–1091.

    Article  Google Scholar 

  • Yamagoe S, Yamakawa Y, Matsuo Y, Minowada J, Mizuno S and Suzuki K (1996) LECT2: Purification of novel human neutrophil chemotactic protein. Immunol. Lett. 52: 9–13.

    Article  PubMed  CAS  Google Scholar 

  • Yamagoe S, Akasaka T, Uchida T, Hachiya T, Okabe T, Yamakawa Y, Arai T, Mizuno S and Suzuki K (1997) Expression of a neutrophil Chemotactic protein LECT2 in human hepatocytes revealed by immunochemical studies using polyclonal and monoclonal antibodies to a recombinant LECT2. Biochem. Biophys. Res. Commun. 237: 116–120.

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto K, Miyoshi-Koshio T, Utuki Y, Mizuno S and Suzuki K (1991) Virucidal activity and viral protein modification by myeloperoxidase: A candidate for defence factor of human polymorphonuclear leukocytes against Influenza infection. J. Infec. Dis. 164: 8–14.

    CAS  Google Scholar 

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Authors
  1. Mie Ito
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  2. Satoshi Yamagoe
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  3. Kazuo Tomizawa
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  4. Satoshi Mizuno
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  5. Masaru Tanokura
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  6. Kazuo Suzuki
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Ito, M., Yamagoe, S., Tomizawa, K. et al. Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli. Cytotechnology 25, 235–238 (1997). https://doi.org/10.1023/A:1007976103088

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  • DOI: https://doi.org/10.1023/A:1007976103088

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