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Prokaryotic Expression,Purification and DNA Binding Activity of DEK Protein’s Carboxyterminal DNAbinding Region |
HUA Ying1 HU Hong-gang1 PENG Xiang-lei |
College of Life Sciences and Bioengineering,School of Science,Beijing Jiaotong University,Beijing100044,China |
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Abstract DEK protein’s carboxyterminal DNAbinding region (CBD) is a newly found DNAbinding domain of DEK,which contains several phosphorylation sites and has a close correlation with DEK protein's function in vivo and in vitro.Using prokaryotic expression system,the peptide of DEK protein's carboxyterminal DNAbinding region (CDB) was expressed and purified.In detail,the CDB DNA fragment was constructed into pET30a (+) vector,and E.coli BL21 (DE3) competent cells were used as host cells.The fusion protein HisCBD was expressed by induction of IPTG and purified by NiNTA agarose.The result of SDSPAGE showed that the molecular weight of the purified protein was about 10.7kDa.Electrophoretic mobility shift assay (EMSA) indicated that DEKCDB prefered to bind to supercoiled form of DNA in vitro,it had similar character to the binding of whole length DEK protein with DNA.This suggested that the carboxyterminal DNAbinding region of DEK protein might function on the binding of DEK protein to DNA partly.
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Received: 16 April 2009
Published: 28 July 2009
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