Expression And Purification Of Human Perforin In Sf9 Insect Cells

Objective:Recent years,how perforin interact with the cell membrane and CTLs(Cytotoxic T Lymphocytes)to kill the target cells has been reported and discovered by many scientists.Perforin plays an important role in the pathogenesis of Hemophagocytic Lymphohistiocytosis(HLH),leukemia,lymphoma,infectious diseases,and autoimmune diseases.It has been proposed that the disease can caused by the imbalance or mutation of perforin in body.Targeted drugs can be studied against perforin,so as to reduce the use of immunosuppressive agents and improve the living quality of patients.However,most of the commercialized perforin proteins are purified by yeast protein expression system,which has no biological activity.The purification of bioactive perforin proteins has been a significant factor restricting the development of perforin-related researches.The purpose of this study is to optimize the method of purifying perforin protein,and to gain the bioactive human perforin protein in more simple and economical way,and to provide offers and new ideas for the subsequent mechanism research of perforin-related diseases.Methods:(1)In order to ensure the yield and activity of the purified perforin,insect cells were used for expression,and the gene sequence of human perforin carrying the label was homologous recombined with the vector,which was transfected into insect cells sf9;(2)To purified the proteins in a simple way,we took a simple and artificial nickel column device for protein affinity chromatography and molecular sieve for concentration.The purified product was concentrated by ultrafiltration tube;(3)In order to determine whether the purified product is human perforin or not,we used coomassie bright blue staining to analyze the product in each step of the purification process,and silver staining method was used to quantify the purified protein.Westtern blot was used to detect the perforin expression and Flag label of the purified protein.(4)In order to measure the biological activity of the purified human perforin,we co-incubated the purified human perforin with human breast cancer cell MCF-7 cells and then adopted propidium iodide staining.The biological activity was analyzed by flow cytometry.Results:(1)We amplified the labeled perforin gene sequence and successfully homologous recombined with the vector,which was transfected into insect cells sf9 and baculovirus was obtained;(2)From the results of coomassie blue staining,we can see that the protein with high purity was purified by a simplified purification method;(3)According to the results of Western blot and sliver staining,the purified product was perforin proteins with His tag and Flag tag,whose purity and production still can be improved;(4)In the presence of Ca2+,the purified perforin protein has the biological activity of pore-formation on human breast cancer cell memberane.Conclusions:The potential for high levels of recombinant protein expression is a major advantage of insect systems,another advantage of insect systems is the ability to process eukaryotic proteins,including the ability to modify proteins such as phosphorylation or glycosylation,insect cells can grow fast,its production is more than eukaryotic system.We can transfect the constructed plasmids into insect cells to obtain baculovirus and store it,the next purification of protein can be completed by taking out baculovirus to infect insect cells,which is more convenient than eukaryotic system and yeast system.In this study,a convenient and rapid method was developed to express and purify bioactive human perforin protein in sf9 insect cells.