[Expression of epidermal growth factor-adenovirus E4orf4 fusion protein in tumor cells and its cytotoxicity].
OBJECTIVE
Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation. EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells. Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells. Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells.


METHODS
EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR. EGF-E4orf4 fragment was cloned into the yeast expression vector. Recombinant plasmid DNA was transformed into the yeast Pichia pastoris. Fusion proteins were purified by SP Sepharose ion exchange chromatography. Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry.


RESULTS
The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4. Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes. Apparent molecular weight of fusion protein was 20 ku. Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody. EGF-E4orf4 in high concentrations (5, and 0.5 microg/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls. Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 microg/3 x 10(5) cells.


CONCLUSIONS
Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.
Bu
Peng-Li
PL
National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100-005, P.R. China.
Wang
Dong-Mei
DM
Chen
Hong
H
Huang
Bing-Ren
BR
D017504:Adenovirus E4 Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001932:Brain Neoplasms / Q000473:pathology*; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D005260:Female; D005910:Glioma / Q000473:pathology; D006801:Humans; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D017504:Adenovirus E4 Proteins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor