%0 Journal Article
%T Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography
%A CHEN Changzheng
%A XIA Qichang
%A LI Boliang
%A WANG Yinglai
%A <br>陈长征
%A 夏其昌
%A 李伯良
%A 王应睐
%J 中国科学C辑(英文版)
%D 1997
%I Springer
%X Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typical (R)RXS/T sequence motif. It suggested that the new method of using ferric (Fe 3 ) chelation affinity chromatography to identify the substrate specificity of protein kinase from random peptide library was feasible.
%K protein kinase
%K enzyme substrate
%K phage display
%K peptide library
%K metal ion chelation affinity chromatography<br>
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=180CF3A72E750F3261A8A60EDC957784&aid=C2D8CAB3656484650018CEF10B827DD1&yid=5370399DC954B911&vid=1371F55DA51B6E64&iid=0B39A22176CE99FB&sid=798FBE8DE1A255B1&eid=23104246A5FCFCEF&journal_id=1674-7305&journal_name=ScienceChina.Lifesciences&referenced_num=0&reference_num=0