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:: Volume 14, Issue 2 (summer 2014) ::
J Ardabil Univ Med Sci 2014, 14(2): 107-117 Back to browse issues page
Evaluating Gene Expression, Purification and Structural Characterization of the Calprotectin Subunits of S100 A8and S100 A9
Hamideh Asghari, Nematollah Gheibi, Kourosh Goodarzvand Chegini , Mahdi Sahmani, Darioush Ilghari
Abstract:   (6502 Views)

   Background & objectives: Calprotectin, S100A8 and S100A9 are involved in important processes including cell signaling and regulation of inflammatory responses. In this study, recombinant expression, purification and structural characterization of S100A8 and S100A9 were accomplished.

  Methods : In this experimental study, pET15b was used as vector of human S100A8 and S100A9 coding sequences, hosted by E.coli BL21 (DE3). Gene expression and purification attempts were evaluated using SDS-PAGE. Protein purification was accomplished using Ni-NTA resin based on its affinity for His-tag present on recombinant proteins. Tertiary structure of proteins were evaluated using spectrofluorimetry.

  Results : The subunits were over expressed 3-4 hours following induction at 37 °C. S100A9 was expressed mainly as inclusion body while S100A8 was found to be expressed mainly as a soluble protein. Purification of S100A8 and S100A9 was achieved at 100 mM imidazole. Spectroscopic studies showed that the amino acid tryptophan is in the internal structures and is less exposed to the aqueous environment.

  Conclusion : In this study, a recombinant S100A8 and S100A9 subunits were expressed and purified and also their structures were confirmed.

 

Keywords: S100A8, S100A9, E.coli BL21(DE3), pET15b, Ni-NTA, Spectroflourimetry
Full-Text [PDF 249 kb]   (1749 Downloads)    
Type of Study: article | Subject: Special
Received: 2013/12/6 | Accepted: 2014/05/15 | Published: 2014/06/30
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Asghari H, Gheibi N, Goodarzvand Chegini K, Sahmani M, Ilghari D. Evaluating Gene Expression, Purification and Structural Characterization of the Calprotectin Subunits of S100 A8and S100 A9. J Ardabil Univ Med Sci. 2014; 14 (2) :107-117
URL: http://jarums.arums.ac.ir/article-1-639-en.html


Volume 14, Issue 2 (summer 2014) Back to browse issues page
مجله دانشگاه علوم پزشکی اردبیل Journal of Ardabil University of Medical Sciences
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