Monitoring Yields Of Active Inclusion Bodies Using The GFP Folding Variants

Compared with the traditional inclusions,active inclusion bodies(AIBs)are easy to be purified,and less poisonous to the host cells.Moreover,AIBs can exert bioactivity without renaturation process.Although it has been reported that a variety of proteins can form AIBs,the folding mechanisms of AIBs is still less clear.Green fluorescent protein(GFP)is one of the proteins that used for detecting AIBs.Recently,various GFPs with multiple folding have been obtained by point mutation of amino acid and related protein screening process,which provides conditions for the research of the protein folding mechanism of AIBs.In this study,we utilized four kinds of GFP variants,such as Em GFP,Se GFP,cp GFP,sf GFP,with different folding to construct recombinantion proteins.and comparative analysis the yields of the AIBs with four kinds of protein mutants which were fused with ELK16,and then proved the correlation of protein folding with yields and activity of the AIBs.The results are as follows:1.The Em GFP ?Se GFP?cp GFP and sf GFP were purified by Ni-NTA affinity chromatography.After then,we used SDS-PAGE to detect a main belt and removed tag by enzyme.Finally,we obtained the target proteins after repurification.2.UV-visible light spectra showed that the maximum absorption at 485 nm.The fluorescence spectra showed that excitation maximum was at 485 nm and the emission maximum was at 510 nm.All purified GFP variants displayed similarly of relative fluorescence intensity with increased concentrations of denaturants such as urea and guanidine hydrochloride,the relative fluorescence intensity was decreased.Among them,the cp GFP was most sensitiveto both denaturants.3.Fusion of ELK16 to the GFP variants,SDS-PAGE and Western blot assay showed that the expression of protein was mainly in insoluble precipitation.The AIBs existed in particular area of cells by laser confocal microscopy.Fourier infrared spectrum analysis showed that the ?-sheet of GFP in AIBs was integrity.4.After induced at 28 ?for 12 h or 37 ? for 4 h,the yields of inclusion bodies cp GFP>Em GFP>Se GFP>sf GFP,which indicated that the yields of inclusion bodies were inversely proportional with the folding.Under different inducing condition,the fluorescence After induced at 28?for6?8?10?12h and 37?for 2?3?4?5h respectively,we analyzed the changes of fluorescence value of recombinant cells.Data dipalyed that the fluorescence intensities of Em GFP and cp GFP were both weaker than Se GFP,which suggested that the mutations of amino acid residues and cycle affected protein folding in the inclusions.The yields of inclusion bodies were high,but the activity was decreased.In summary,we used four kinds of GFP folding variants to investgate relationship between the yields and the protein productivity and activity in the AIBs.This was consistent with the relationship between the soluble protein expression and function reported previously.Thus,it still faces challenges to get the ideal AIBs with high yields and activity.The constructed system is potentially appled for used to screen efficient aggregating tags for transforming the target protein into AIBs.