Design, Production, and Characterization of Recombinant Kedarcidin Apoprotein in Escherichia coli

• Main Authors: Jen-Sheng Chiu, 邱仁昇
• Other Authors: Der-Hang Chin
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/77707079498004918653

碩士 === 國立中興大學 === 化學系 === 93 === Enediyne chromoproteins are potent antitumor antibiotic agents. Kedarcidin (KDC) belongs to this category and is composed of two components, an acidic single chain polypeptide, KDC apoprotein (apo-KDC), and an enediyne containing chromophore (KDC-chr). The apo-KDC tightly binds to and highly stabilizes the labile KDC-chr. Cleavage of histone protein by apo-KDC was also demonstrated in the past. Recently, the proteolytic property of another enediyne chromoprotein, neocarzinostatin, is disproved using a tool of recombinant apoprotein. Owing to the functional significance of apo-KDC in the drug performance and the interesting puzzle in its proteolytic property, in the current investigation we design and characterize a recombinant apo-KDC in Escherichia coli using a ligation-independent cloning method. An artificial gene for KDC apoprotein (apo-KDC) production in Escherichia coli is designed and constructed on a pCAL-n-EK vector. Six complementary overlapping oligonucleotides are annealed by PCR method to generate a 373-base pair double-stranded DNA fragment. The vector pCAL-n-EK is treated with Eam1104 I and is gel-purified. To produce single-stranded overhangs, the digested pCAL-n-EK vector is incubated with Pfu polymerase in the presence of dTTP. To produce complementary overhangs on the insert DNA, the artificial gene PCR product is also treated with the same polymerase in the presence of dATP. The vector and the insert DNA are then annealed at room temperature and transformed directly into E. coli DH5α competent cells. The CBP-tagged apo-KDC protein is purified and enterokinase is used to remove the CBP tag followed by final purification using DEAE ion exchange chromatography. The obtained apo-KDC is identified using SDS-PAGE, UV, HPLC and LC-MS analyses.