Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Science in China. Series C, Life Sciences
C ChenY Wang

Abstract

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 displaying it on the surface of filamentous bacteriophage fd. Phosphorylationin 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-rner peptide library phosphorylated by cAPK was selected with ferric (Fe(3+)) chelation 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. Theirin 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 chromamraphy to identify the substrate specificity of protein kinase from random peptide library was feasible.

References

Jan 1, 1991·Methods in Enzymology·B E KempC M House
Sep 1, 1990·Trends in Biochemical Sciences·B E Kemp, R B Pearson
Apr 1, 1986·Analytical Biochemistry·L Andersson, J Porath
Feb 1, 1995·Current Opinion in Biotechnology·R CorteseM Sollazzo
Mar 12, 1993·Cell·Z SongyangR J Lechleider
Jan 1, 1993·Methods in Enzymology·G P Smith, J K Scott
Dec 1, 1994·Current Opinion in Structural Biology·E J Goldsmith, M H Cobb
Jul 19, 1994·Proceedings of the National Academy of Sciences of the United States of America·R J GoodsonS Rosenberg

❮ Previous
Next ❯

Related Concepts

Related Feeds

Bacteriophage: Phage Therapy

Phage therapy uses bacterial viruses (bacteriophages) to treat bacterial infections and is widely being recognized as an alternative to antibiotics. Here is the latest research.

Related Papers

Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica
Jia-Da LiKe-Yi Wang
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica
Chang-Zheng ChenYing-Lai Wang
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
Yan XuanChao Liang
© 2022 Meta ULC. All rights reserved