Recovery of recombinant zebrafish p53 protein from inclusion bodies and its binding activity to p53 mRNA in vitro.
p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. Soluble p53 protein with high purity was successfully obtained using the optimized renaturation approach. Results of a UV-crosslinking experiment and immunoprecipitation:RT-PCR analysis confirmed that the purified p53 protein could bind specifically to its cognate mRNA. Our results suggest that selecting a suitable buffer is important for renaturing p53 protein from inclusion bodies. We also demonstrated a specific interaction between p53 and it own RNA in zebrafish. Measurement of the binding activity may be a useful approach for identifying the activity of recombinant p53 protein in vitro.
Zhao
Xiangzhong
X
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Graduate School, Chinese Academy of Sciences, Beijing 100039, China.
Liu
Ming
M
Wu
Ning
N
Ding
Lili
L
Liu
Haizhou
H
Lin
Xiukun
X
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D029961:Zebrafish Proteins