Rhodococcus sp. RHA1 유래의 Indole Oxygenase의 클로닝 및 발현
Cloning and Expression of Indole Oxygenase Gene Derived from Rhodococcus sp. RHA1
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An indole oxygenase originated from Rhodococcus sp. RHA1 was cloned into the expression vector,pTrc99A, in Escherichia coli, and designated pTCAN1. The pTCAN2 was constructed from pTCAN1 by the deletion of lacIq for the constitutive expression of indole oxygenase without adding IPTG in the medium. The complete open reading frame of indole oxygenase was 1,224 bp long, which encodes a protein of 407amino acids. Crude extracts of E. coli DH5α/pTCAN1 and pTCAN2, respectively, were prepared and subjected to SDS-PAGE analysis. A band corresponding to molecular mass of about 43 kDa was appeared and this result correlated with the predicted molecular mass of cloned indole oxygenase. The E. coli harboring pTCAN1 and pTCAN2, respectively, showed blue color colony in LB plate. The pigment showing blue color was prepared from E. coli DH5α/pTCAN2, and identified as indigo by experiments using spectrophotometer, HPLC, and TLC. The indigo-forming activity of indole oxygenases from the whole cell of E. coli DH5α/pTCAN1 cultured at LB medium added 1mM of IPTG and that of E. coli/pTCAN2 showed about 1.75nmol/min/mg DCW (dry cell weight) and 3.85 nmol/min/mgDCW, respectively. Also, the E. coli DH5α/pTCAN2 produced about 236 μM of indigo after 48 hours incubation in TB medium supplemented with 2.5 mM of tryptophan.
Abstract
An indole oxygenase originated from Rhodococcus sp. RHA1 was cloned into the expression vector,pTrc99A, in Escherichia coli, and designated pTCAN1. The pTCAN2 was constructed from pTCAN1 by the deletion of lacIq for the constitutive expression of indole oxygenase without adding IPTG in the medium. The complete open reading frame of indole oxygenase was 1,224 bp long, which encodes a protein of 407amino acids. Crude extracts of E. coli DH5α/pTCAN1 and pTCAN2, respectively, were prepared and subjected to SDS-PAGE analysis. A band corresponding to molecular mass of about 43 kDa was appeared and this result correlated with the predicted molecular mass of cloned indole oxygenase. The E. coli harboring pTCAN1 and pTCAN2, respectively, showed blue color colony in LB plate. The pigment showing blue color was prepared from E. coli DH5α/pTCAN2, and identified as indigo by experiments using spectrophotometer, HPLC, and TLC. The indigo-forming activity of indole oxygenases from the whole cell of E. coli DH5α/pTCAN1 cultured at LB medium added 1mM of IPTG and that of E. coli/pTCAN2 showed about 1.75nmol/min/mg DCW (dry cell weight) and 3.85 nmol/min/mgDCW, respectively. Also, the E. coli DH5α/pTCAN2 produced about 236 μM of indigo after 48 hours incubation in TB medium supplemented with 2.5 mM of tryptophan.
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