Super Folder Green Fluorescent Protein Mediate Recombinant Fusion Protein Extracellular Secretion In Escherichia Coli.

As a new type of report gene,GFP has been widely used in almost all fields of biology research.Super folder GFP(sfGFP)as a robust fold type GFP which is well folded unless the fusion protein,when the target protein fused to sfGFP,it could obtain a better folding and solubility,especially when the silently solve and dissolve protein fused to sfGFP.On the conditional opportunity,we found that sfGFP could be detected in the culture medium when it expressed in the E.coli strain Rosetta Blue,and under the regulation of the T7 promoter without IPTG induction.Aiming at this phenomenon,we made preliminary research on the secretion mechanism of sfGFP,and explored a simple and universal platform for the extracellular secretion of heterologous protein in E.coli based on the extracellular secretion characteristic of sfGFP.The main points of this research are summarized as follows:1.Detection of sfGFP extracellular secretion phenomenon and preliminary research on the extracellular secretion mechanism of sfGFP.When sfGFP expressed in the E.coli strain Rosetta Blue,meanwhile,at the same culture condition,the super charged Green fluorescence protein(ScGFP)expressed as a positive control and expression vector pET23a-T(designed by our lab)expressed as a negative control,we found that sfGFP protein could secrete into the culture medium and be detected by the SDS-PAGE and Western-blot analysis,while the ScGFP protein could not be detected in the culture medium.Aiming at this phenomenon,we made a preliminary research on the extracellular secretion mechanism of sfGFP,and draw the following conclusions:sfGFP secretion is not due to the cell lysis during the whole culture process,sfGFP extracellular secretion is a kind of independent phenomenon,which did not rely on any exist classical secretion pathway.From the result of this research,we can take a conclusion that:sfGFP secretion process could be divided into three steps:first step,synthesis sfGFP in the cytoplasm trans-located from the inner membrane into the periplasmic space;second step,sfGFP in the periplasmic space anchored into the outer-membrane;third step,sfGFP released from the outer membrane and secrete into the culture medium.The net negative charges provide sfGFP a high soluble level and major force,well folded ?-barrel structure provide sfGFP insert into the membranes(inner-and outer-membranes)and combined with the cytoplasmic membrane mobility,in which sfGFP could trans-locate form the cytoplasm into the periplasmic space,anchor into the outer-membrane and release from the outer-membrane and extracellular secrete into the culture medium.2.Fusion protein extracellular secretion in E.coli mediate by sfGFP.In this research,based on the extracellular secretion characteristic of sfGFP,we constructed a simple and universal vector for heterologous protein gene clone and extracellular express in E.coli,in which the heterologous protein fused to the C-terminal of the sfGFP and expressed as a fusion form.The coding sequence of target gene was added to the 3' terminal of sfGFP gene by the endogenous homologous recombination technology in E.coli to construct the expression vector.A series of polypeptides and proteins have been fused to the C-terminal of sfGFP and expressed in E.coli,and we found that sfGFP could mediate polypeptides,enzyme(?-N-acetylaminoglucoside enzyme H),multi-metric protein(human arginase 1),Agon protein(glutamic acid decarboxylase),cytoplasm membrane trans-location,outer-membrane trans-location and extracellular secretion.As a fusion tag,sfGFP did not affect the activity of fused protein,fusion protein sfGFP-ARG1,sfGFP-GAD,sfGFP-Endo H,still remained their activity.3.High cell density fermentation of sfGFP fusion protein in E.coli.In this research,fed-batch fermentation method was used for the high cell density fermentation in E.coli.The whole process were described as follows:first step,the feeds were cultured at 37? in SOB shaking flask;second step,when the OD600 reach at 1.5,the feeds were transferred into fermentation medium(SOB,add MgCl2 at final concentration of 0.1mol/L)and cultured at 37?;third step,when the fermentation reach the exponential growth point,supplementary medium(200 g/L yeast extract,100 g/L glycerol,15 g/L of alpha lactose)began to add and cultivation temperature dropped to 30? for the induction,forth step,the fed-batch fermentation process was stopped when the fusion protein had the highest extracellular secretion level.Most sfGFP fusions have been obtained an improved extracellular secretion level,when used the fed-batch fermentation method,fusion protein sfGFP-ARG1 obtained the highest secretion level of 0.94 mg/mL,with the activity of 299.25 U/mg.4.Purification and digestion of sfGFP fusion protein and purification of target protein.In this research,6×His tag was add to the N-terminal of sfGFP to facilitate the purification step of sfGFP fusions,between the sfGFP and target protein,an TEV enzyme digestion site was add for the rapid cutting off sfGFP fusions and releasing the target protein.The results show that the purity of sfGFP reached more than 90%after a Ni-NTA affinity purification step.After rapid digested by proteinase(Proteinase TEV,28? 5 h),and the target protein released from the sfGFP fusion.With a one-step NI-NTA purification of the proteinase production to remove the sfGFP tag,more than 92%purity target protein was obtained.