Insertional-fusion of basic fibroblast growth factor endowed ribonuclease 1 with enhanced cytotoxicity by steric blockade of inhibitor interaction |
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Authors: | Tada Hiroko Onizuka Masayuki Muraki Kazuko Masuzawa Wataru Futami Junichiro Kosaka Megumi Seno Masaharu Yamada Hidenori |
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Institution: | Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan. tadahrk@biotech.okayama-u.ac.jp |
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Abstract: | Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase. |
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