Prokaryotic Expression And Physicochemical Properties Analysis Of Vanderwaltozyma Polyspora Wss1

In normal cells,there is a certain amount of DNA-protein crosslinks?DNA-protein cross-links,DPCs?,which is the result of normal DNA and nuclear protein interaction or metabolism and play an important role in maintaining the normal cell activities.Cells can be induced to produce excess DPCs when subjected to such as ionizing radiation,formaldehyde,other physical and chemical factors,which can block helicase,thereby preventing DNA replication and subsquent cell division.Wss1 metalloproteinases as a rare DPCs repair enzymes was currently found that the protein element of DNA-protein crosslinks can be cleavaged in order to make organissms accurately copy their genetic information even in the case of cross-linking.Meanwhile,Wss1 has a unique character,which has a DNA-dependent,that means its self-cleavage can be activted only in the case of DNA-containing.This indicates that the enzyme has a special responsibility,which can remove the genome crosslinked in order to protect the stability of the genome.So far,reports on metalloproteinases Wss1 is little to know and its action mechanism is unclear.In this study,we constructed prokaryotic expression vector of the Vanderwaltozyma polyspora Wss1 and got the protein of VpWss1 by prokaryotic expression and purification.The protein homogeneity,aggregation status,and DNA-dependent self-cleavage activity were analyzed,which not only provides basis for the further research on the structure and function of metalloproteinase Vp Wss1,but also provided in-depth reference for the study on its action mechanism.The main conclusions of this study are summarized as follows:1.In this study,we synthesised codon-optimized Vanderwaltozyma polyspora Wss1 gene and constructed a recombinant plasmid pET-15b?BE-?SUMO-VpWss1,which N-terminal containing 6 His,SUMO double tags.Then transformed the plasmid into Escherichia coli host strain 2566.The fusion protein is a soluble induced by 0.3mmol / L IPTG,18?,16 h and its molecular weight is about 50 kD.2 We compared the gene sequences of Saccharomyces cerevisiae Wss1 with Vanderwaltozyma polyspora Wss1,designing appropriate primers and mutanted Glu96 to Gln96 using the method of overlap extension PCR amplification,eventually got the mutant VpWss1^E96Q.3.The fusion protein was crude separated by His Trap^TM FF affinity column and AKTA Prime plus protein purification,then further separated by SP.Finally,we got the wild-type and mutant VpWss1 metalloproteinase with purity greater than 95%.4.The homogeneity of wild-type and mutant protein were analyzed and compared on the liquid state by dynamic laser light scattering?DLS?and gel filtration chromatography.The results showed that wild-type VpWss1 exist with poor uniformity,showing a variety of states,the homogeneity of mutant protein was significantly improved compared with wild-type,the major component of the single state appeared to be more than 93%.This mutant can be used as an important material for the subsequent crystallization and structural analysis.5.By analyzing the influences of different DNA to the self-cleavage activity of Wss1.We observed that the WCE,ssDNA and dsDNA can induce the self-cleavage of metalloproteinase Wss1.Moreover,the self-cleavage induced by 44nt-ssDNA and 36bp-dsDNA were stronger,51nt-ss DNA and 45bp-ds DNA were weaker.However,from the results of self-cleavage activity,we found that the mutant Wss1^E96Q was less affected by the different length of DNA,and the self-cleavage activity was significantly reduced compared with the wild type.In the study,Wss1 and the mutant Wss1^E96Q were expressed and purified successfully,further their homogeneity,self-cleavage activity were clarified.These results laid the foundation for the crystal preparation and structure analysis.