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Updated: Oct 13, 2025
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational
Iqra Ishrat1,2, Aoxing Cheng1,2, Fazhi Yu2
1Department of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China.
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.
