Expression And Purification Of Proliferating Cell Nuclear Antigen In Insect Cells

Proliferating cell nuclear antigen(PCNA)is a cofactor of DNA polymerase ?,playing essential roles in DNA replication,DNA repair,chromatin structure maintenance and cell-cycle progression.PCNA is identified to be an important antigen associated with the autoimmune diseases-systemic lupus erythematosus(SLE).So far,less studies have reported the recombinant expression and purification of PCNA.In this work,the full length recombinant human PCNA protein was produced in a baculovirus expression system by Spodoptera frugiperda(Sf9)and BTI-TN-5B1-4(High 5)insect cells.Several factors were adjusted to optimize the expression,including multiplicity of infection(MOI)value and infected time,and the optimal condition was MOI 0.05,infection time 6 d.The content of rPCNA protein obtained from the cell lysate and culture supernatant from Sf9 cells was similar and after one step of Ni-NTA affinity chromatography,they were both less than 2 mg/L,both with a purity only 70%.However,high expression of rPCNA protein was achieved within High 5 cells.The yield of rPCNA protein was about 100 mg/L High 5 cell lysate with a purity>90%after one-step Ni-NTA affinity chromatography.And rPCNA further purified by strong anion exchange chromatography(Q sepharose),the final yield of the rPCNA was about 70 mg/L cell lysate,with a purity>95%.Immunogenic activity tests by indirect ELISA demonstrated that the purified rPCNA protein obtained high biological activity,with the sensitivity and specificity of 93.3%and 85.0%,respectively.Together,this work established an expression and purification procedure for recombinant human PCNA protein,which will have potential applications on diagnosis of PCNA-associated diseases in vitro.