Cloning and expression of the Pseudomonas aeruginosa Ifo 3455 alkaline proteinase and the determination of the active site of the enzyme by site-directed-mutagenesis

Harteni D., . and Noor Aini Abdul Rashid, . and Morihara K., . (1995) Cloning and expression of the Pseudomonas aeruginosa Ifo 3455 alkaline proteinase and the determination of the active site of the enzyme by site-directed-mutagenesis. [Proceedings Paper]

The 4.1kb alkaline proteinase gene on the 7.0kb Sau 3A fragment has been successfully subcloned into the pUC 18 vector and transformed into Escherichia coli JM109. Here expression was achieved only after the isolation bodies and refolding of the enzyme in vitro. However the expression could no longer be observed after several subculturing stages and it was extremely difficult to subculture. The gene had to be transformed into another strain DH5alpha. Here the gene had gone through deletions which probably prevented the expression of the gene from under the lac promoter. It could be interpreted that the gene product could be relatively toxic to E. coli and E. coli uses its built-in defensive mechanism to modify the gene. Therefore another strain of E. coli was used. In this strain ABLE C appeared stable. This is probably because the strain is able to regulate copy numbers and this could maintain the expression level low enough therefore it was less deleterious to E. coli

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Abstract

The 4.1kb alkaline proteinase gene on the 7.0kb Sau 3A fragment has been successfully subcloned into the pUC 18 vector and transformed into Escherichia coli JM109. Here expression was achieved only after the isolation bodies and refolding of the enzyme in vitro. However the expression could no longer be observed after several subculturing stages and it was extremely difficult to subculture. The gene had to be transformed into another strain DH5alpha. Here the gene had gone through deletions which probably prevented the expression of the gene from under the lac promoter. It could be interpreted that the gene product could be relatively toxic to E. coli and E. coli uses its built-in defensive mechanism to modify the gene. Therefore another strain of E. coli was used. In this strain ABLE C appeared stable. This is probably because the strain is able to regulate copy numbers and this could maintain the expression level low enough therefore it was less deleterious to E. coli

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Item Type:	Proceedings Paper
Additional Information:	Summary En
AGROVOC Term:	PSEUDOMONAS AERUGINOSA
AGROVOC Term:	CLONACION MOLECULAR
AGROVOC Term:	EXPRESION GENICA
AGROVOC Term:	ESCHERICHIA COLI
AGROVOC Term:	PROTEASAS
Geographical Term:	MALAYSIA
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:26
URI:	http://webagris.upm.edu.my/id/eprint/15383

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