Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Mutation of Angiogenesis Inhibitor TK1-2 to Avoid Antigenicity In Vivo

  • Lee Sang-Bae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) ;
  • Kim Hyun-Kyung (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) ;
  • Oh Ho-Kyun (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) ;
  • Hong Yong-Kil (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea) ;
  • Joe Young-Ae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
  • Published : 2006.03.01
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Abstract

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

Keywords

References

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