PMID- 15841157
OWN - NLM
STAT- MEDLINE
DCOM- 20061212
LR  - 20201209
IS  - 1671-167X (Print)
IS  - 1671-167X (Linking)
VI  - 37
IP  - 2
DP  - 2005 Apr 18
TI  - [Isolation and identification of Comamonas testosteroni: cloning and 
      overexpression of 3alpha-hydroxysteroid dehydrogenase in E.coli].
PG  - 203-6
AB  - OBJECTIVE: To isolate and identify 3alpha-hydroxysteroid dehydrogenase 
      (3alpha-HSD) producing Comamonas testosteroni from soil, and to clone and 
      overexpress 3alpha-HSD in E.coli. METHODS: Samples of pond mud were inoculated 
      into cultural medium with androsterone as sole carbon source. The primary 
      identification was performed according to the morphological observation, 
      biochemical reaction and cultural characterization. To further identify the 
      bacteria, a couple of primers were designed according to the 3alpha-HSD gene of 
      Comamonas testosteroni. An 800 bp fragment containing 3alpha-HSD gene was 
      obtained by PCR amplification. Then the PCR products were inserted into plasmids 
      pET-15b to construct recombinant plasmids pET-15b. Afterwards the host bacteria 
      containing recombinant plasmids pET-15b with proper orientation grew with 
      isopropyl-beta-D-thioglactopyranoside (IPTG) induction. RESULTS: The isolated 
      bacteria which could use androsterone as the sole carbon source had 85% 
      consistency with Comamonas testosteroni. After 5 hours of IPTG induction, a 
      recombinant protein about 29 x10(3) with enzyme activity was overexpressed in the 
      host bacteria E.coli. BL21(DE3) pLysS. This protein could catalyze the 
      dehydrogenization reaction of androsterone (3alpha-hydroxysteroid). CONCLUSION: A 
      strain of Gramjnegative 3alpha-HSD producing Comamonas testosteroni was isolated 
      from pond mud, and recombinant 3alpha-HSD with enzyme activity was overexpressed 
      in E.coli. This work laid good foundation for the purification of recombinant 
      3alpha-HSD by metal chelate chromatography, and also for the construction of an 
      enzymatic cycling method to measure serum total bile acids with recombinant 
      3alpha-HSD as the tool enzyme.
FAU - Zhang, Guo-hua
AU  - Zhang GH
AD  - Department of Clinical Laboratory Medicine, Peking University First Hospital, 
      Beijing 100034, China.
FAU - Cong, Ai-ri
AU  - Cong AR
FAU - Xu, Guo-bin
AU  - Xu GB
FAU - Sun, Li-ying
AU  - Sun LY
FAU - Yan, Yan
AU  - Yan Y
FAU - Xia, Tie-an
AU  - Xia TA
LA  - chi
PT  - Journal Article
PL  - China
TA  - Beijing Da Xue Xue Bao Yi Xue Ban
JT  - Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health 
      sciences
JID - 101125284
RN  - 0 (Recombinant Proteins)
RN  - EC 1.1.1.50 (3-alpha-Hydroxysteroid Dehydrogenase (B-Specific))
SB  - IM
MH  - 3-alpha-Hydroxysteroid Dehydrogenase 
      (B-Specific)/biosynthesis/*genetics/isolation & purification
MH  - Cloning, Molecular
MH  - Comamonas testosteroni/*enzymology/*isolation & purification
MH  - Escherichia coli/genetics/*metabolism
MH  - Recombinant Proteins/biosynthesis/genetics
EDAT- 2005/04/21 09:00
MHDA- 2006/12/13 09:00
CRDT- 2005/04/21 09:00
PHST- 2005/04/21 09:00 [pubmed]
PHST- 2006/12/13 09:00 [medline]
PHST- 2005/04/21 09:00 [entrez]
PST - ppublish
SO  - Beijing Da Xue Xue Bao Yi Xue Ban. 2005 Apr 18;37(2):203-6.