Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 194-202.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0465

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Expression and Purification of the MLL3SET Protein with a Site-directed Mutation of an Unnatural Amino Acid

WANG Xiao-qin1(), HUANG Yin-ping1, WANG Wei-qian2, WU Ping2, QUAN Shu1()   

  1. 1. Nactional Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237
    2. Nation Facility for Protein Science in Shanghai,Zhangjiang Lab,Shanghai Advanced Research Institute,Chinese Academy of Sciences,Shanghai 201210
  • Received:2021-04-09 Online:2022-03-26 Published:2022-04-06
  • Contact: QUAN Shu E-mail:wxq870718522@163.com;shuquan@ecust.edu.cn

Abstract:

This work aims to introduce the unnatural amino acid,N-propargyl-lysine(PrK),into the catalytic domain(MLL3SET)of histone H3K4 methyltransferase MLL3,to express and purify the mutant protein(MLL3SET*),and evaluate its enzyme activity,which will lay the foundation for further single-molecule fluorescence resonance energy transfer(smFRET)experiments to characterize the mechanism of MLL3. MLL3SET was linked into pET-28b(+)to construct an expression vector. Residue N4905 was selected for PrK introduction upon MLL3SET crystal structure analysis. The commercial E.coli strain(C321. ΔA. exp)was genetically modified to integrate the T7 RNA polymerase gene. Further,MLL3SET* was expressed and purified in the modified strain,and finally the enzyme activity was determined. The results showed that the constructed C321. ΔA. exp lacZT7p07 strain harboring the pET28b-MLL3SET* plasmid normally expressed the target protein after co-transformation with the aaRS-tRNA orthogonal system and the addition of PrK. The MLL3SET* was purified successfully by Ni-NTA affinity chromatography and gel filtration chromatography. The results of the multi-component enzyme-coupled reaction showed that although the enzyme activity of MLL3SET* was lower than that of wild-type MLL3SET,it retained about 43% of the wild-type enzyme activity. This study successfully achieves prokaryotic expression and purification of PrK labeled MLL3SET protein retaining certain enzymatic activity,which lays a foundation for the subsequent in-depth study of the molecular mechanism of MLL3SET.

Key words: MLL3SET, genome modification, unnatural amino acid, expression and purification, enzyme activity