Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines.
OBJECTIVE
To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.


METHODS
Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.


RESULTS
Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.


CONCLUSIONS
This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.
Dai
Li-Cheng
LC
Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, Huzhou 313000, Zhejiang Province, China. dlc@hzhospital.com
Xu
Di-Yong
DY
Yao
Xing
X
Min
Li-Shan
LS
Zhao
Ning
N
Xu
Bo-Ying
BY
Xu
Zheng-Ping
ZP
Lu
Yong-Liang
YL
D001943:Breast Neoplasms; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D008297:Male; D000078792:Midkine; D010957:Plasmids / Q000235:genetics; D011471:Prostatic Neoplasms; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D016207:Cytokines; D011993:Recombinant Fusion Proteins; D000078792:Midkine; D049452:Green Fluorescent Proteins