Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli.
Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.
Nozaki
Seishiro
S
Department of Pharmaceutical Sciences, Tohoku University Hospital and Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.
Tomioka
Yoshihisa
Y
Hishinuma
Takanori
T
Inoue
Masayuki
M
Nagumo
Yoko
Y
Tsuruta
Lilian R
LR
Hayashi
Katsuhiro
K
Matsumoto
Toshiki
T
Kato
Yoshinori
Y
Ishiwata
Shunji
S
Itoh
Kunihiko
K
Suzuki
Toshio
T
Hirama
Masahiro
M
Mizugaki
Michinao
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001059:Apoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D053281:Enediynes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism; D005454:Fluorescence Polarization; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013950:Thymus Gland / Q000737:chemistry; D009353:Zinostatin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001059:Apoproteins; D053281:Enediynes; D006657:Histones; D011994:Recombinant Proteins; C052401:aponeocarzinostatin; C032372:neocarzinostatin chromophore; D009353:Zinostatin; D004996:Ethidium