Expression Of Recombinant Human Kallistatin Protein In Chinese Hamster Ovary Cells And Its Anti-hepatoma Study

Kallistatin(KAL)is a multifunctional protein with anti-inflammatory,antioxidative stress,anti-fibrotic,anti-angiogenic,anti-tumor and other functions.Our group previously used yeast engineering strain to manufacture recombinant KAL protein.However,titer of the monoclonal antibody prepared by KAL protein from yeast engineering strain was still considerably poor.It might be due to changes in the molecular conformation of the KAL protein.Therefore,here we mainly constructed efficient expression vector of CHO-K1 cells and selected for stable CHO-K1-KAL strains instead.After serum-free acclimation,KAL protein was expressed and purified.We investiagted the effect of KAL in liver cancer at the end.The methods and results of this study are presented as follows:Firstly,18 kinds of vectors including Kozak sequence,human albumin signal peptide,syntheticly designed signal peptide and cell-penetrating peptide TAT with pcDNA3.1,pTT5,pMH3,pMH3 EN vectors were constructed by genetic engineering.Transient transfection and sandwich ELISA were applied to select the vector with the highest expression of KAL protein.Secondly,the highest expression vector was transfected into CHO-K1 cells in optimal transfection conditions.After transfection,the cells were digested and transferred to culture dishes.G418 was used to select the positive strain,single colonies were picked and subcultured into 96-well plates,and G418 was further used to select.After 7 days,the ELISA was used to detect the KAL concentrations of cell supernatant to select the positive strain at absorbance value of above 1.0.The limiting dilution method was used for cloning.After three times of cloning,the culture was gradually expanded and the stable strain expressing KAL was successfully selected.Acclimated suspending serum-free,optimized batch expression,KAL protein was purified by Ni column,and identified by SDS-PAGE and Western blot.Finally,The effect of KAL on hepatoma cells by CCK-8,Hoechst 33342 staining and transwell experiment were carried out,and the possible mechanism of KAL on anti-liver cancer by Western blot was tested.The results of this study showed that different signal peptides and cell-penetrating peptides were of different effects on the secretion and expression of proteins.The syntheticly designed signal peptide used for this study has significantly increased the expression of KAL in different vectors.The KAL expression level of the optimal vector constructed in this study was about 10 times higher than that of the unoptimized vector.Consequently,four stable strains of CHO-K1 expressing high level concentration of KAL protein,i.e.2F8,4B4,4E7 and 7D11,were selected and the highest concentration of 11 mg/L KAL protein was obtained after 10 days.The molecular weight of recombinant KAL protein expressed in CHO-K1 cells was about 60 kD subjected to purification by batch expression of Ni column.It was slightly larger than that of E.coli(40 kD)and yeast(58 kD).This might be due to its glycosylation modification and 6×His tag.At the end,we found that KAL could induce apoptosis of liver cancer cells and inhibit hepatoma cell migration and invasion.The better understanding of how KAL inducing hepatoma cells apoptosis is required further exploration.