The Inhibitory Effect Of Zinc Ions On The Self-cleavage Of Intein Npu And The Effect Of Intermolecular Disulfide Bonds On The Trans-cleavage

Background and aim:Inteins,as a functional protein that can excise themselves from precursor proteins and concomitantly ligate the two exteins together with a peptide bond,it has been recognized and exploited rapidly in the past ten years.With the further research of the splicing mechanism,the application of inteins got more and more extensive.For example,its application in protein purification which the inclusion of inteins enable to remove the tag from the purification systems easily and avoid the use of protease,in protein connection,in gene diagnosis and treatment,biosensor,and so on.In the early stage,there are many continuous inteins,such as Sce VMA,mini-Mtu RecA,Ssp DnaB and so on.They make a lot of contribution in the process of purification and connection of proteins.But the main drawback was the uncontrollable premature cleavage during the expression of the recombinant protein.After the artificial fracture of Ssp DnaB was discovered and used,the possibility of uncontrollable cleavage was reduced by separating the two fragments of intein.Npu DnaE is a kind of natural occurred split intein,which can directly solve the problems mentioned above.Unfortunately,such purification requires two protein expression systems with a high cost,which will not be a good choice in industrial amplification.Therefore,we try to inhibit the cleavage of inteins by zinc ions which will solve the problem of uncontrollable premature cleavage.The result further expands the application of the inteins in the host cells,and provides an immense idea for industrial amplification and production,especially for the production in the field of biological drugs.Mutation of Npu D118G inhibited N-terminal cleavage and significantly improve C-terminal cleavage effiency.In the past,the addition of DTT is the necessary condition to induce the reaction of inteins since the presence of disulfide bonds caused by cysteines.We engineered a kind of mutation of Npu,was no longer dependent on the addition of reducing agent,such as DTT,which has implications for designing of thiol-independent C-cleavage DnaE inteins and for understanding the structural and dynamical trais of DnaE inteins.Method:In this study,Escherichia coli BL21?DE3?was selected as the host cell and the inhibition of zinc ions was explored by using the natural split intein-Npu DnaE.The N-terminal and C-terminal of Npu DnaE were respectively combined with the human IgG-Fc and PE38KDEL who have many cycstine residues.Co-express the fusion proteins and add 1mM².5mM zinc ions.Inhibition of the self-splicing effiency can be detected by SDS-PAGE and Western-blot.The concentration of zinc ions in E.coli.was measured by ICP.The C-terminal cleavage of activity of Npu_D118G was used to investigate the formation of intermolecular and intramolecular disulfide bonds under non-reducing and reducing conditions.Through overlap-PCR and site-directed mutation clone of the C1A,C28S,C59S and C+1A,mutation plasmids got constructed and then expressed and purified by Ni-column.Whether the cleavage activity of the inteins with no cycsteine residues remained under different temperatures was independent of the presence of DTT was detected by SDS-PAGE.Results:The two segments of the intein Npu DnaE were fused into the target protein to form‘Fc-NpuN'and‘Npu C-PE'.After treated the two fusion proteins with 1mM².5mM Zn²⁺,it was found that the growth status of E.coli.was not affected,the expression of Fc-NpuN was not affected and the expression of NpuC-PE was not affected.When co-expressed the two plasmids,after intein Npu DnaE got self-cleavage,the new fusion protein“Fc and PE”significantly decreased with the growing concentration of Zn²⁺.The results showed that the self-cleavage of Npu DnaE could be inhibited effectively by adding Zn²⁺in E.coli.which provide an experimental basis for expanding the application of inteins used in the production of biological drugs.In the process of studying the cleavage effiency of Npu DnaE caused by disulfide bonds,it was confirmed that the existence of cysteines formed intermolecular and intramolecular disulfide bonds.And the cleavage efficiency was no longer affected by DTT induced in the mutations of cysteine residues to serine.The same conclusion can be obtained under 4?,22?,37?temperature conditions.This method can be used to design and construct a kind of thiol-independent C-terminal cleavage DnaE inteins.Meanwhile,it provides an understanding of the structural and dynamical traits of DnaE inteins.