TY  - JOUR
T1  - Cloning, Expression and Purification of Nanog Protein from <I>Capra hircus</I> in <I>Escherichia coli</I>
AU - Zheng, X.B. AU - Cen, X.M. AU - Wang, L.X. AU - Xin, G.Y. AU - Fu, X.H. AU - Liu, P. AU - Li, G.H. AU - Zhang, M. AU - Lu, S.S. AU - Lu, K.H. 
JO  - Journal of Animal and Veterinary Advances
VL  - 12
IS  - 2
SP  - 201
EP  - 207
PY  - 2013
DA  - 2001/08/19
SN  - 1680-5593
DO  - javaa.2013.201.207
UR  - https://makhillpublications.co/view-article.php?doi=javaa.2013.201.207
KW  - Capra hircus
KW  -Nanog gene
KW  -cloning
KW  -prokaryotic expression
KW  -protein purification
AB  - Nanog is one of important transcription factors to maintain 
  characteristics of pluripotent stem cells. The present study was to clone Nanog 
  of <I>Capra hircus</I> to express His-Nanog protein in <I>E. coli</I> BL 21 
  cells and further to purify it. The total RNA was extracted from primordial 
  genital ridge tissues of a fetal lam and by means of RT-PCR, <I>Nanog</I> gene 
  was amplified which was subcloned to pET32a to construct its prokaryotic expression 
  vector. Confirmed by restrictive endonuclease digestion and DNA sequencing, 
  the recombinant plasmid was transformed into <I>E. coli</I> BL21(DE3) and His-Nanog 
  fusion protein was expressed by the induction of IPTG and identified with SDS-PAGE 
  analysis. Under denaturing condition, the His-Nanog protein was purified by 
  using Ni-NTA resin and verified by Western blotting assay. The results showed 
  that The Open Reading Frame (ORF) of <I>Nanog</I> gene in <I>Capra hircus</I> 
  is composed of 903 nucleutide acids, coding 320 amino acids; SDS-PAGE assay 
  showed that His-Nanog fusion protein was efficiently expressed in form of inclusion 
  bodies in <I>E. coli</I> BL21 (DE3) inclusion bodies were solubilized in 6 mol 
  L<SUP>-1</SUP> GuHCl, His-Nanog fusion protein with higher purity was purified 
  by using Ni-NTA resin; Western blotting assay showed that the purified His-Nanog 
  could bind to anti-His tag antibody specifically indicating the expected immunogenicity. 
  This recombinant protein could be used directly to prepare polyclonal or monoclonal 
  anti-Nanog antibody which will lead to study Nanog&#146;s function or characteristics 
  of pluripoten stem cells (such as iPS cells) in <I>Capra hircus</I>.
ER  -