Biol. Pharm. Bull. 29(5) 1010—1014 (2006)

tumor chromoprotein antibiotics for liver cancer, stomach cancer, bladder cancer, acute leukemia, isolated from culture filtrates of Streptomyces carzinostaticus var. F-41 (S. carzinostaticus). NCS is composed of two components, and acidic single chain polypeptide (apo-NCS) and a labile, ninemembered ring enediyne-containing chromophore (NCSchr). Because NCS has several problems in clinical cases, the SMANCS (conjugate of NCS and poly (styrene-comaleic acid anhydride)) was developed by Maeda and his co-workers for the treatment of protopathic liver cancer. And Okamoto and his co-workers were developed the A7-NCS for the treatment on peritoneal dissemination of gastric cancer, which is a conjugate of a monoclonal antibody against the human stomach cancer cell line A7 and NCS. Thus, some modifications are performed in the apoprotein part of the chromoprotein in the functional improvement because the apo-NCS is proposed to work as a packing-carrier protein to stabilize and carry the biological active NCS-chr. Therefore, the apo-NCS is the natural drug delivery system (DDS), and its apoprotein will be a promising pro-protein for the creation of the man-made drug binding protein given the ability of the unnatural function. Recently, Heyd et al. demonstrated the good approach in the creation of drug delivery vehicle using apo-NCS derived random phage-display library, called “in vitro evolution” of apo-NCS. And proteins reactive with testosterone derivatives were prepared. This report also supports above our idea that the man-made drug binding protein derived from apo-NCS in described before. The strategy of the single or several site-specific mutagenesis is effective for functional analyses for the amino acid side chain on the apo-NCS. We reported here that W39F, F52Y, S98A, S98G, and S98C were newly prepared and investigated their physicochemical properties. We have reported the importance of Phe78 using a single site-specific mutant F78W. And we also investigated the circular dichroism (CD) spectral analysis of F78W, because it remains whether this change of EtdBr binding to F78W is derived from the unstabilization of protein structure or steric hindrance of Trp. Moreover, the randomized library of apo-NCS on the phage display system was prepared using megaprimer PCR mutagenesis and overlap extension PCR mutagenesis with dam methylase methylation/DpnI digestion.