Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity

Yoshihisa Tomioka, Shigeki Kisara, Saki Yoshizawa, Mika Ozawa, Naoto Suzuki, Hiroaki Yamaguchi, Takanori Hishinuma, Michinao Mizugaki, Junichi Goto

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 μM for wild type, 2.2 μM for S98A, 1.3 μM for S98G, 9.7 μM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.

Original languageEnglish
Pages (from-to)1010-1014
Number of pages5
JournalBiological and Pharmaceutical Bulletin
Volume29
Issue number5
DOIs
Publication statusPublished - 2006 May

Keywords

  • Ethidium bromide
  • Fluorescence polarization analysis
  • Neocarzinostatin
  • Phage display
  • Randomized library

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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