Soluble expression of native EGFP in a cell-free translation system

Objective: To construct the prokaryotic expression plasmid that can express enhanced green fluorescent protein(EGFP) in a cell-free translation system.Methods: Utilizing plasmid as template,full-length EGFP gene was amplified.The pEGFP-N1 gene digested with NcoI,HindⅢ was ligated to pET28a vector and digested with the same restriction enzymes,then the resultant ligation product was transformed into E.coli DH5α.Positive clones were propagated and recombinant plasmids were extracted for further identification and sequencing.The sequence-confirmed recombinant plasmid was then given the name,pET28a-EGFP.The macroprepared pET28a-EGFP(0.6mg/ml) was added into the Cell-Free translation system,and incubated for 3 hours.The fluorescence was observed by the fluorescence microscopy.EGFP was identified by Western blotting. Results: DNA sequencing confirmed that the EGFP gene sequence in pET28a-EGFP was in accordance with that of GenBenk.The intensive fluorescence in cell-free translation system was seen under the fluorescence microscope.Western blotting demonstrated that expressed protein of about 27000 was native EGFP. Conclusion: The pET28a-EGFP which can efficiently express the soluble native EGFP in cell-free translation system was successfully constructed,which provided a basis to further establish a standard substance of fluorescence intensity.