[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].
OBJECTIVE
To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity.


METHODS
The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis.


RESULTS
The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity.


CONCLUSIONS
Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.
Yue
Chen-Li
CL
Respiratory Division of Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Shi
Jie-Ran
JR
Shi
Chang-Hong
CH
Zhang
Hai
H
Zhao
Lei
L
Zhang
Ting-Fen
TF
Zhao
Yong
Y
Xi
Li
L
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D016982:Micrococcus luteus / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000254:growth & development
D001426:Bacterial Proteins; D016207:Cytokines; C113704:resuscitation-promoting factor, bacteria