Construction And Functional Characterization Of A Recombinant Metarhizium Robertsii CPD Photolyase

Ultraviolet(UV)radiation can cause many damages in humans,including skin darkening and photoaging,as well as epidermal damage including acute sunburn and hyperpigmentation.At the cellular level,the main DNA damage caused by UV radiation are two photoproducts,cyclobutane pyrimidine dimers(CPD)and pyrimidine-pyrimidinone(6-4)photoproducts(6-4PPs).CPD damage is the main cytotoxic lesion of UV radiation-induced cell death,and human cells remove CPD from nuclear DNA with a complex repair system.However,CPD photolyase exists in microorganisms,plants and a few animals.CPD enzyme can directly repair CPD on DNA after activated by visible light,which is a simple and efficient way.In order to to repair CPD on human nuclear DNA with CPD photolyase,this study create a CPD recombinant photolyase based on microorganisms and the only known CPD photolyase from mammals to automatically enter the human nucleus to repair UV damages.The main research results are as follows:1)We verified that AE1(the third helix of the Antennapedia homeodomain translocates protein)and TE1(the 49-60 amino acid of the HIV-trans transcription activator protein)were both cell-penetrating peptides.It has the function of transmembrane transport that can carry the recombinant protein into human cells.On this basis,by screening the nuclear signal peptides,cell penetrating peptides and target proteins,a recombinant protein structure model(SVNLS-target protein-AE1)that can transport exogenous proteins into cells across cell membrane and nuclear membrane was constructed,where SVNLS is the nuclear localization signal of simian virus 40.2)According to the recombinant protein structure model(SVNLS-target protein-AE1)obtained above,we constructed the recombinant protein expression vector of CPD photolyase from long-nosed kangaroo and Metarhizium Robertsii which is widely distributed in nature.However,the recombinant protein SVNLS-Pt PL-AE1 of the CPD photolyase(Pt PL)from the long-nosed kangaroo could not be expressed in E.coli,but the recombinant protein SVNLS-Mr PHR1-AE1 of the CPD photolyase(Mr PHR1)from Metarhizium Robertsii was successfully expressed.The optimal expression and purification conditions of the recombinant protein SVNLS-Mr PHR1-AE1 were obtained by optimization:the temperature of inducting protein expression was 10℃～20℃;the buffer(including lysis buffer,wash buffer and elute buffer)used in purification process was p H 7,and the salt ion concentration was 300 m M Na Cl.3)It was found that the CPD content in the nuclear DNA of human skin fibroblasts HFF-1 increased by about 150 times after 1J/m~2 UV irradiation,and then there was no significant change after visible light irradiation;The CPD content on nuclear DNA of cells incubated with SVNLS-Mr PHR1-AE1 recombinant protein was decreased by 35%after irradiating UV and visible light(activation of SVNLS-Mr PHR1-AE1 protein).4)In addition to directly acting on the DNA of cells and causing DNA damage such as CPD,UV can also induce cells generate reactive oxygen species(ROS)to further induce DNA damage.ROS can also act on unsaturated fatty acids in lipids to generate lipid peroxides,and the latter can be gradually decomposed into a series of complex compounds including the highly toxic molecule malondialdehyde(MDA)which poisons cells.We found that the level of intracellular ROS increased by 2.3 times and the level of MDA increased by 5.4 times in ordinary HFF-1 cells after UV irradiation.The ROS content of cells incubated with SVNLS-Mr PHR1-AE1recombinant protein after UV treatment decreased to a level comparable to that of cells without UV treatment after white light irradiation,while the MDA content also decreased by 2-fold.5)As an oxygen free radical scavenger,superoxide dismutase(SOD)can effectively scavenge intracellular ROS to protect cells.The intracellular SOD activity of HFF-1 cells increased by 2.7 times after irradiating UV,while the intracellular SOD activity of cells incubated with SVNLS-Mr PHR1-AE1 recombinant protein was further increased after irradiating visible light which was up to 4 times more than the cells without UV treatment.The ability of recombinant protein SVNLS-Mr PHR1-AE1 to increase SOD enzyme activity in UV-treated cells may be one of the reasons why it can reduce the level of ROS and MDA in such cells.In conclusion,this study successfully constructed,expressed and purified the recombinant protein SVNLS-Mr PHR1-AE1 of CPD photolyase from Metarhizium Robertsii.The recombinant protein can cross the cell membrane and nuclear membrane and enter the nucleus of human cells HFF-1 to clear the CPD damage of nuclear DNA induced by UV radiation,and can effectively reduce the levels of oxygen free radicals and toxic substance malondialdehyde induced by UV,and increased SOD enzyme activity in UV-irradiated cells.Therefore,the recombinant protein SVNLS-Mr PHR1-AE1 based on fungal CPD photolyase has good application value in repairing and preventing UV damage to human cells,and provides an effective method for bringing exogenous proteins into the nucleus of human cells.