Prokaryotic Expression Of Recombinant KLK7 And Its Activity Research In Pancreatic Cancer Cells

Background According to domestic and foreign research and the previous research of the research group,kallikrein-related peptidase 7(KLK7)can promote the proliferation,metastasis and invasion of pancreatic cancer cells,and inhibition,silence,knockdown and knockout of KLK7 can reduce the proliferation,metastasis and invasion ability of pancreatic cancer cells,and can also lead to Ferroptosis in pancreatic cancer cells.Objective In this study,the active KLK7 protein was expressed and purified by prokaryote,and replenished into the pancreatic cancer cells with KLK7 deficiency through replenishment experiments,and its effect on the function of pancreatic cancer cells was explored,which provided scientific basis for KLK7 as a potential therapeutic target for pancreatic cancer.Methods(1)The prokaryotic expression vectors of p GEX-6P-1-KLK7 were constructed by molecular cloning technology.(2)The recombinant KLK7 protein was expressed by prokaryotic expression system.(3)GST-KLK7 was purified with GST affinity magnetic beads and activated with enterokinase.(4)The KLK7 activity was identified with fullwavelength fluorescent enzyme marker and fluorescent substrate.(5)The active function of KLK7 active protein in pancreatic cancer cells was verified by rescue experiment: the effect of KLK7 on the proliferation of PANC-1KD/KO cells was evaluated by CCK8 method,realtime cell detection technology and clone formation experiment,and its effect on migration and invasion was evaluated by scratch test and Transwell test;The effect of KLK7 protein on the proportion of living cells in pancreatic cancer cells was analyzed by live cell count;Western blot were used to analyze the reversal effect of KLK7 active protein on iron death like changes caused by KLK7 deficiency.Results(1)The p GEX-6P-1-KLK7 prokaryotic expression vector was successfully constructed.(2)The prokaryotic expression system was successfully used to express GSTKLK7 recombinant protein,and GST-KLK7 recombinant protein was purified by GST affinity magnetic beads,and KLK7 protein was obtained by shear activation.(3)Supplementation of KLK7 protein into PANC-1 cell lines of pancreatic cancer cells with KLK7 deficiency can significantly improve the proliferation,metastasis and invasion ability of pancreatic cancer cells.(4)Replenishment of KLK7 protein can reverse the Ferroptosis caused by KLK7 deficiency.Conclusion This study showed that the expression of active KLK7 protein by prokaryotic can significantly improve the proliferation,metastasis and invasion of PANC-1 in pancreatic cancer cells with KLK7 deficiency.And it can reverse Ferroptosis caused by KLK7 deletion,providing scientific basis for KLK7 as a potential therapeutic target for pancreatic cancer.