Basic Research
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 21, 2005; 11(31): 4812-4814
Published online Aug 21, 2005. doi: 10.3748/wjg.v11.i31.4812
Screening and identification of proteins interacting with nucleostemin
Hai-Xia Yang, Geng-Lin Jin, Ling Meng, Jian-Zhi Zhang, Wen-Bin Liu, Cheng-Chao Shou
Hai-Xia Yang, Geng-Lin Jin, Ling Meng, Jian-Zhi Zhang, Wen-Bin Liu, Cheng-Chao Shou, Department of Biochemistry and Molecular Biology, Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing 100034, China
Author contributions: All authors contributed equally to the work.
Supported by the National High Technology Research and Development Program of China, No. 200BA711A11A06 and Beijing Science and Technology Project, No. H020220020310
Correspondence to: Dr. Cheng-Chao Shou, Department of Biochemistry and Molecular Biology, Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing 100034, China. cshou@vip.sina.com
Telephone: +86-10-66160960 Fax: +86-10-66175832
Received: December 25, 2004
Revised: January 23, 2005
Accepted: January 26, 2005
Published online: August 21, 2005
Abstract

AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS.

METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially.

RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A.

CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

Keywords: Nucleostemin, Yeast two-hybrid, Co-IP