A Highly Soluble Green Fluorescent Protein Expressing In Escherichia Coli

With the rapid advance of genomics,bioinformatics and proteomics,various protein’s functions remain to be identified.Fusion protein tagging technology has been applied to protein localization,protein-protein inter66 action and purification etc.At present,many fusion labeling systems are widely utilized in the expression of recombinant proteins.However,these techniques are laborious,cost-effective and complicated.An ideal protein tag would be genetically encoded,work both in vivo and in vitro,provide a sensitive analytical signal and not interfere with or even enhance the solubility of target protein.In prokaryotic expression system,most expressed product of wild-type Green Fluorescent Protein(wtGFP)are insoluble and prone to form inclusion bodies.As a reporter gene,it can be widely used in gene expression and regulation,protein localization,transfer and interaction,signal transduction,transfection and transformation,cell separation and purification.Its folding is readly affected by temperature,and the luminescence is weak.It is also not easy to be observed in some multilayer plant cells.The expressed product of modified enhanced GFP(eGFP)is still partial soluble in prokaryotic expression system.If eGFP is used as a label,it is not conducive to the study of the structure,function and biochemistry of soluble recombinant protein.In this study,Green Fluorescent Protein(GFP)was used to express soluble recombinant protein in prokaryotic expression system.And the strategies of soluble expression of recombinant protein in E.coli were optimized: the vector,the host,changing the culture parameters of the recombinant host strain,and optimizing the gene sequence.In this study,according to the preference of codon-usage in E.coli and the characteristic that GFP was mutated for some amino acids that can form superfolding structure,and thereupon the nucleotides encoding 16 amino acids were modified.The expression of synthesized soluble GFP(sGFP)in E.coli strain of BL21(DE3),was conducted using eGFP as a control and vector pET-29b(+)as a blank control.The fluorescence intensity,solubility and soluble expression conditions of sGFP were screened.The fluorescence intensities of eGFP and sGFP were detected by NightSHADE LB 985 in vivo.In the range of isopropyl-β-D-thiogalactoside(IPTG)concentration of 0~20 μm,the in vivo average fluorescence intensity of sGFP was always stronger than that of eGFP.When the concentration of IPTG used was 15 μm,the fluorescence intensities of both eGFP and sGFP were the strongest,and the in vivo fluorescence intensity of sGFP was 2.897 times higher than that of eGFP.Therefore,the optimal induction concentration of IPTG was determined to be 15 μm.In order to further detect the high fluorescence characteristics and solubility of sGFP,protein expression was induced under optimal induction conditions,and the supernatants and precipitates were analyzed after ultrasonication.The results showed that the sGFP supernatants was yellow under visible light and the eGFP supernatants was yellowish.Under the ultraviolet excitation light of 360 nm,sGFP displayed green fluorescence stronger than that of eGFP,and the fluorescence intensity of its supernatants was significantly higher than that of the precipitates.The green fluorescence of eGFP was not obvious,and the fluorescence intensity of the precipitates was slightly higher than that of the supernatants.In order to further demonstrated the high fluorescence intensity of sGFP,sGFP and eGFP expression were induced in vivo.The results showed that the single colony fluorescence intensity of sGFP was stronger than that of eGFP,which could be visualized under visible light.The supernatants and precipitates were analyzed by NightSHADE LB 985,and the fluorescence intensities of sGFP and eGFP were significantly different.The results showed that the average fluorescence intensity in the sGFP supernatants was 21.93 times higher than that of the precipitates,and was 29.05 times higher than the average fluorescence intensity of eGFP in the supernatants.The analytical data showed that sGFP has high fluorescence characteristics,and it has also been shown that the expression of sGFP in E.coli is highly soluble.To further verify the high solubility of sGFP,total protein,supernatants and precipitates were analyzed by SDS-PAGE electrophoresis.Using the Tanon GIS software to calculate the gray density value of the photo protein spots in gel,the results showed the solubility of sGFP was stronger than that of eGFP,in which sGFP and eGFP were 82.72% and 41.25% respectively by comparing supernatants with precipitates.In order to develop an indicator tool that is simple to operate and determines the percent solubility of the fusion target protein based on the unit of fluorescence measurement.Purified sGFP was utilized.The concentration of sGFP was determined by BCA method,and the result showed that the sGFP concentration was 458.5 ng/μL.And the quantitative correlations between the fluorescence intensity of sGFP and its concentrations were established.The results showed that the fluorescence intensity(cps)of sGFP showed a good linear relationship with its mass concentration(ng/uL).When the concentration of sGFP was lower than 0.8 ng/μL,the fluorescence intensity was still detected,which proved its high sensitivity.It will lay a foundation for evaluating the soluble expression concentration of the fusion protein.This method is simple to evaluate the solubility and concentration of the fused protein.This assay also provides a sensitive,time-saving and feasible tool to analyze the signal for fusion protein expression,protein localization,weak protein interaction and so forth.It also furnishes with a prove of principle for the development of soluble protein tagging system.