Expression, purification, and characterization of soluble RpfD with high bioactivity as a recombinant protein in Mycobacterium vaccae.
Resuscitation promoting factor D (RpfD) is one of the five members of the Rpf-like family in Mycobacterium tuberculosis, which all have the resuscitation-promoting activity. Here, the complete rpfD gene was amplified and cloned into pDE22 expression vector. Then a recombinant form of soluble RpfD (sRpfD) was expressed in Mycobacterium vaccae. The soluble recombinant RpfD was purified by Ni-Sepharose affinity chromatography. The purified final product was >95% pure, and the molecular weight was 24.0kDa, determined by 15% SDS-PAGE stained with Coomassie brilliant blue R-250. The yield of purification was about 1.10mg/L of the culture. The biochemical property of the sRpfD was analyzed by stimulating the resuscitation of avirulent M. tuberculosis H37Ra which was in "non-culturable" condition. The results indicate that the sRpfD from M. vaccae could more efficiently stimulate the resuscitation of M. tuberculosis H37Ra than the refolded recombinant RpfD (iRpfD) from Escherichia coli DH5alpha, and that the rabbit anti- sRpfD serum could completely inhibit this resuscitation-promoting effect caused by these two kinds of recombinant RpfD. Our study indicates that this expression system may facilitate large-scale production and purification of sRpfD which have high biological activity for further functional, pharmacological and clinical investigations.
Gao
Hui
H
Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, 710032, Shaanxi, PR China.
Bai
Yinlan
Y
Xue
Ying
Y
Wang
Limei
L
Fan
Ailing
A
Ding
Xin
X
Xu
Zhikai
Z
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D012995:Solubility
D001426:Bacterial Proteins; D016207:Cytokines; D007106:Immune Sera; D011994:Recombinant Proteins; C113704:resuscitation-promoting factor, bacteria