Investigation Of Split-GFP System To Detect Peptide Fragment Insertion Proteins

Peptide insertion is an effective method to investigation the relationship between protein folding,structure and function.The Split-GFP reporting system can effectively monitor the effect of linker peptide insertion on the solubility and folding of target protein in Escherichia coli.Split-GFP including the C-terminal 15-amino-acid fragment of the GFP(GFPC),and 215-amino acid fragment of non-fluorescent GFP(GFPN)emits fluorescence after complementation in vivo and in vitro.It can be used to evaluate the expression of inserted GFPC in target proteins that affect GFPN self-assembly in vitro and in vivo.Fluorescence complementary technology can be used to detect the effect of protein soluble expression and folding damage on protein structure and function;Peptide insertion allows certain proteins to be expressed as inclusion bodies and used as an aggregation tag to confer new functions on the protein.1)Vector construction: The sequence of GFPC was inserted into the specific position of MBP and GST of the solution-promoting tag by overlapping PCR and phosphorylation connection technology,and the His6-tag was fused before the tag for purification of the target protein.GFPC sequences were added at specific sites of β-lactamase to detect changes in Ampicillin tolerance.2)Several recombinant plasmids were co-expressed with pACYC-GFPN in E.coli BL21(DE3)competent state.From the results of SDS-PAGE and WB It was found that peptide insertion affects protein water solubility and impairs folding,making it expressed as inclusion bodies.3)The supernatant protein spectrum scanning results showed that the fluorescence spectroscopy properties didn’t change,and the absorption peak didn’t shift,which laid the foundation for the detection of fluorescent protein as an indicator protein in the later period.Flow cytometry detection of bacterial liquid fluorescence showed that different tags had different effects on the expression of bacterial liquid fluorescence.4)The results of the bacterial confocal laser microscopy examination showed that the insertion of the peptide into the GST tag would affect the folding of the protein in the body and achieve aggregated expression.The same result is found in MBP,and it is more serious than GST.5)Peptide insertion severely reduces the efficiency of the tag protein to bind the affinity resin.The binding capacity with Ni-NTA decreased slightly(About 24%reduction)and the insertion of peptide fragments changed the conformation and folding of the targeted protein,Thereby affecting the purification efficiency of the tag.6)Fluorescent protein was fused at the C-terminus to detect the solubilization function of the protein.From the fluorescence measurement results,the solubilization effect of the tagged protein did not change significantly.7)Limited hydrolysis is the most effective method for detecting protein foldability.The supernatants of several purified recombinant proteins are reacted with trypsin.The results show that peptide insertion reduces protein stability and is more easily hydrolyzed by trypsin at 30℃.After hydrolysis,the internal GFPC is released.Which self-assembles with GFPN in vitro to generate fluorescence,and the same result is also produced in the MBP-Tag.8)The insertion of a GFPC sequence into β-lactamase reduced the resistance of the strain to Ampicillin.