A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells.
Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahistidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by two-dimensional gel electrophoresis (2DE) and identified by MALDI-TOF MS/MS. A total of 27 protein spots including glucose-regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells.
Kim
Jee Yon
JY
Dept. of Biological Sciences, KAIST, 373-1, Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Kim
Yeon-Gu
YG
Baik
Jong Youn
JY
Joo
Eun Jin
EJ
Kim
Young Hwan
YH
Lee
Gyun Min
GM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D004921:Erythropoietin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D023822:Tissue Engineering
D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin