[Study on the fusion expression of FBXO30: a novel member of F-box protein family].
The cDNA sequence of the coding region of FBXO30 (F-box only protein 30), which is a novel member of F-box protein family, was cloned into the mammalian expression vector pEGFP-C2 by non-directional cloning method and introduced into the NIH 3T3 cells by liposome transfection. Observation under the fluorescent microscopy after transfection showed that EGFP (enhanced green fluorescent protein)/FBXO30 was expressed and existed mainly in the cytoplasm. At the same time, the cDNA sequence of the coding region of FBXO30 was cloned into prokaryotic expression vector pGEX-4T-2 by directional cloning strategy. The GST (glutathione S-transferase)/FBXO30 fusion protein was expressed under the induction of IPTG (isopropylthio-beta-D-galactoside) in E. coli. A new band (approximately 65.5 kD) was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Purification of the GST-fused FBXO30 was carried out by affinity chromatography with glutathione sepharose 4B. The fusion expression of FBXO30 indicates that: FBXO30 protein is a cytoplasmic soluble protein, and the stable fusion expression of FBXO30 in the prokaryotic expression system can provide a base for the preparation of the specific antibody of FBXO30 and further functional study of FBXO30.
Li
Z H
ZH
Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, China.
Zhang
X H
XH
Li
G Y
GY
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics; D050980:GATA Transcription Factors; D005982:Glutathione Transferase / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D050980:GATA Transcription Factors; D018398:Homeodomain Proteins; D015514:Oncogene Proteins, Fusion; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C103965:srp protein, Drosophila; D005982:Glutathione Transferase