• Journal of Life Science
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• v.11 no.2
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• pp.108-110
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• 2001

The E. coli expression system to overproduce a harmful protein, carboxypeptidase Taq was developed. Since expression plasmid pCK305N containing the colicin promoter already has the initiation codon on the restriction site, the initiation codon of the CPase Taq gene was removed. Expression plasmid pCP4-col includes the entire CPase Taq gene, which is directed by the colicin promoter. E. coli cells harboring pCP-col produced a high amount of the enzyme when they were cultured in the present of mitomycin C (0.4 ${\mu}g$/ml). An amount of purified enzyme produced by pCP4-col directed by the colicin promoter was 10.5 mg. This result indicated that the novel E. coli expression system controlled by the colicin promoter could produce almost twice amounts of CPase Taq than the conventional system controlled by the tart promoter.

• Journal of Life Science
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• v.12 no.6
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• pp.682-687
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• 2002

We analyzed improvement on the enzyme activity of CPase Taq by addition of various metal ions. The enzyme activity was increased more then four times by 1 mM cobalt ion and almost three times by 1 mM calcium ion. However, the active center metal zinc ion did not affect the enzyme activity. In order to investigate whether the active center metal affects the enzyme activity, zinc ion which is occupied the active center of the enzyme was replaced by cobalt ion which activates the enzyme activity very effectively. Since the cobalt ion in the active center of the cobalt-substituted CPase Taq did not affect the enzyme activity, it could act as the natal metal ion in the active center of the enzyme.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.389-392
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• 2004

In order to produce a harmful protein more efficiently, this expression cassette, dubbed pCol-MICT, is directed by the colicin promoter, and was constructed by the insertion of a $rrnBT_1T_2$ fragment of pEXP7, and a MxelnteinCBD fragment of pTXB3, into pSH375. To test whether harmful proteins, including proteolytic enzymes, could be effectively produced by this cassette, the carboxypeptidase (CPase) Taq gene was inserted into the pCol-MICT cassette to yield pCol-CPase Taq-MICT. E coli W3l 10 tells harboring pCol-CPase Taq-MICT produced a large quantity of this enzyme, as much as 47.2 mg of purified from per liter of culture, when cultured in the presence of mitomycin C ($0.4{\mu}g/mL$). This indicates that the colicin promoter-controlled E, coli expression cassette was able to produce almost 8 times of protein than the conventional tar promoter-based system, and that this cassette may be useful in the Synthesis of other harmful proteins.

• Proceedings of the Korean Society of Life Science Conference
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• 2002.09a
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• pp.87-87
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• 2002

• Proceedings of the Korean Society of Life Science Conference
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• 2001.09a
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• pp.125-125
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• 2001