Iqra Ishrat1,2, Aoxing Cheng1,2, Fazhi Yu2, Jing Guo3, Peng Zhang4, Kaiguang Zhang5, Zhenye Yang6,7. 1. Department of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China. 2. Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. 3. Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China. 4. Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. peng0912@ustc.edu.cn. 5. Department of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China. zhangkaiguang@ustc.edu.cn. 6. Department of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China. zhenye@ustc.edu.cn. 7. Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. zhenye@ustc.edu.cn.
Abstract
BACKGROUND: Post-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions. METHODS: To overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells. RESULTS: Using this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays. CONCLUSION: Thus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.
BACKGROUND: Post-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions. METHODS: To overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells. RESULTS: Using this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays. CONCLUSION: Thus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.
Authors: Taro Hitosugi; Sumin Kang; Matthew G Vander Heiden; Tae-Wook Chung; Shannon Elf; Katherine Lythgoe; Shaozhong Dong; Sagar Lonial; Xu Wang; Georgia Z Chen; Jianxin Xie; Ting-Lei Gu; Roberto D Polakiewicz; Johannes L Roesel; Titus J Boggon; Fadlo R Khuri; D Gary Gilliland; Lewis C Cantley; Jonathan Kaufman; Jing Chen Journal: Sci Signal Date: 2009-11-17 Impact factor: 8.192