含非天然氨基酸定点突变的MLL3SET蛋白表达与纯化

doi:10.13560/j.cnki.biotech.bull.1985.2021-0465

摘要/Abstract

摘要：

在组蛋白H3K4甲基转移酶MLL3的催化结构域（MLL3_SET）中定点引入非天然氨基酸N-炔丙基赖氨酸（N-propargyl-lysine,PrK）,表达、纯化该突变蛋白（MLL3_SET*）,并评估突变蛋白的酶活,为后续进一步利用单分子荧光共振能量转移技术（smFRET）表征MLL3的作用机制奠定基础。将MLL3_SET接入pET-28b（+）构建表达载体,通过MLL3_SET晶体结构分析选择N4905位点进行PrK的引入;对商业化菌株（C321. ΔA. exp）进行基因组改造以引入T7 RNA聚合酶基因,并在改造后的菌株中表达、纯化MLL3_SET*,最后测定MLL3_SET*的酶活性。结果表明,在构建的C321. ΔA. exp lacZ∷T7p07菌株中,pET28b-MLL3_SET* 在共转入aaRS-tRNA正交系统以及外源添加PrK后能够正常表达;通过Ni-NTA亲和层析及凝胶过滤层析成功纯化出高纯度的MLL3_SET*蛋白;多酶级联反应结果显示,MLL3_SET*的酶活性比野生型蛋白低,但仍具有约43%的甲基转移酶活性。本研究成功实现了含PrK的MLL3_SET 蛋白的原核表达与纯化,突变蛋白保留了一定酶活性,为后续深入研究MLL3的分子机制奠定了基础。

关键词: MLL3_SET, 基因组改造, 非天然氨基酸, 表达纯化, 酶活性

Abstract:

This work aims to introduce the unnatural amino acid,N-propargyl-lysine（PrK）,into the catalytic domain（MLL3_SET）of histone H3K4 methyltransferase MLL3,to express and purify the mutant protein（MLL3_SET*）,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. MLL3_SET was linked into pET-28b（+）to construct an expression vector. Residue N4905 was selected for PrK introduction upon MLL3_SET crystal structure analysis. The commercial E.coli strain（C321. ΔA. exp）was genetically modified to integrate the T7 RNA polymerase gene. Further,MLL3_SET* was expressed and purified in the modified strain,and finally the enzyme activity was determined. The results showed that the constructed C321. ΔA. exp lacZ∷T7p07 strain harboring the pET28b-MLL3_SET* plasmid normally expressed the target protein after co-transformation with the aaRS-tRNA orthogonal system and the addition of PrK. The MLL3_SET* 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 MLL3_SET* was lower than that of wild-type MLL3_SET,it retained about 43% of the wild-type enzyme activity. This study successfully achieves prokaryotic expression and purification of PrK labeled MLL3_SET protein retaining certain enzymatic activity,which lays a foundation for the subsequent in-depth study of the molecular mechanism of MLL3_SET.

Key words: MLL3_SET, genome modification, unnatural amino acid, expression and purification, enzyme activity

王小琴, 黄银萍, 王蔚倩, 吴萍, 全舒. 含非天然氨基酸定点突变的MLL3_SET蛋白表达与纯化[J]. 生物技术通报, 2022, 38(3): 194-202.

WANG Xiao-qin, HUANG Yin-ping, WANG Wei-qian, WU Ping, QUAN Shu. Expression and Purification of the MLL3_SET Protein with a Site-directed Mutation of an Unnatural Amino Acid[J]. Biotechnology Bulletin, 2022, 38(3): 194-202.

图/表 10

表1 PCR反应引物序列

Table 1 Primer sequences of PCR reactions

引物名称 Primer name	序列 Sequence（5'-3'）
Kan-F	TTCGCGTTCGCGTAAGTGTAGGCTGGAGCTGC
Kan-R	TTACGCGAAATACGGGCAGACATGGCCTGCC- CGGTTATTAATATCCTCCTTAGTTCCTATTCC
T7p07-F	GGAATTGTGAGCGGATAACAATTTCACACAGG- AAACAGCTATGAACACGATTAACATCGCTAAG
T7p07-R	CTCCAGCCTACACTTACGCGAACGCGAAGTCC
（T7p07+Kan）-F	GGAATTGTGAGCGGATAAC
（T7p07+Kan）-R	TTACGCGAAATACGGGCAG
F1	CCAGAAAGGAGAAGAGCTCTCCTATGACTAT- AAGTTTGAC
R1	GTCAAACTTA TAGTCATAG GAGAGCTCTTCT- CCTTTCTGG
F2	GAGAAGCTTTATGAGTGTCAGAACCGTGGTG- TGTAC
R2	GTACACACCACGGTTCTGACACTCATAAAGC- TTCTC
F3	GTGGAGCTGTGTAGTGCCGGAAGTGGATG
R3	CATCCACTTCCGGCACTACACAGCTCCAC

图1 多酶级联反应测定MLL3SET*活性的原理图在MLL3SET和AR复合物（M3AR）及其他酶的催化下,SAM经过多步反应,最终生成的物质N-（4-安替比林）-3-氯-5-磺酸酯苯醌-单亚胺在515 nm处具有吸光度

Fig. 1 Diagram of the multi-enzyme coupling reaction for determining MLL3SET* activity Under the catalysis of MLL3SET and AR compound（M3AR）and other enzymes,SAM undergoes multiple reactions. The final product N-（4-antipyryl）-3-chloro-5-sulfonate-ρ-benzoquinone-monoimine shows absorbance at 515 nm

图2 基因组改造表达测试 M：蛋白分子量标准;U：未诱导总菌体裂解液;I：诱导总菌体裂解液

Fig. 2 Expression test after genomic modification M：Protein marker. U：Lysate of uninduced whole cell. I：Lysate of induced whole cell

图3 MLL3SET结构信息（A）和表达质粒图谱（B）将MLL3SET的第4 883位半胱氨酸突变为丝氨酸,将4 819位丝氨酸突变为半胱氨酸,将4 905位的天冬酰胺突变为琥珀密码子。A：MLL3SET六个半胱氨酸的位置信息（PDB：5F6K）,MLL3SET结构为黄色;底物H3为海蓝色;产物SAH、6个半胱氨酸以及2个标记位点用棍棒模型显示;锌离子用灰色小球显示;B：pET28b-His-sumo-mll3set C4883S S4819C N4905X质粒图谱

Fig. 3 MLL3SET structural information（A）and the map of the expression plasmid （B） The cysteine at position 4 883 of MLL3SET is mutated to serine,serine at position 4 819 to cysteine,and asparagine at position 4 905 to the amber codon. A：Structural information of 6 cysteine residues in MLL3SET（PDB：5F6K）. MLL3SET is colored in yellow;the substrate H3 is colored in aquamarine. The product,SAH,6 cysteines,and the two sites chosen for smFRET labeling are displayed as the stick model. The zinc ion is shown in the grey sphere. B：pET28b-His-sumo-mll3set C4883S S4819C N4905X plasmid map

图4 pET28b-His-sumo-mll3set C4883S质粒的表达测试 M：蛋白分子量标准;U：未诱导总菌体裂解液;I：诱导总菌体裂解液;S：诱导裂解液上清;P：诱导裂解液沉淀;37℃：在37℃表达;16℃：在16℃表达

Fig. 4 Expression test of the pET28b-His-sumo-mll3set C4883S plasmid M：Protein marker. U：Uninduced whole cell after lysis. I：Induced whole cell after lysis. S：Induced supernatant after lysis. P：Induced precipitation after lysis. 37℃：Expression at 37℃. 16℃：Expression at 16℃

图5 表达His-SUMO-MLL3SET*分析 A：SDS-PAGE分析;B：Western blot分析. M：蛋白分子质量标准;1-4：宿主菌为C321.ΔA. exp lacZ∷T7;1：未诱导总菌体裂解液;2：诱导总菌体裂解液;3：诱导裂解液上清;4：诱导裂解液沉淀;5-6：宿主菌为BL21（DE3）;5：未诱导总菌体裂解液;6：诱导总菌体裂解液

Fig. 5 Analysis of expressed His-SUMO-MLL3SET* A：SDS-PAGE analysis. B：Western blot analysis. M：Protein marker;1-4：The host strain was E. coli C321.ΔA. exp lacZ∷T7. 1：Uninduced whole cell after lysis. 2：Induced whole cell after lysis. 3：Supernatant of induced cells after lysis. 4：Precipitation of induced cells after lysis. 5-6：The host strain was E. coli BL21（DE3）. 5：Uninduced whole cell after lysis. 6：Induced whole cell after lysis

图6 纯化His-SUMO-MLL3SET*分析 M：蛋白分子质量标准;1：诱导总菌体裂解液;2：诱导裂解液上清;3：诱导裂解液沉淀;4：洗涤前的流出缓冲液;5：用50 mL 缓冲液洗涤后的漂洗液;6：用1 L洗涤缓冲液洗涤后的漂洗液;7：用缓冲液重悬混合均匀咪唑洗脱前的Ni beads;8：咪唑洗脱液;9：用缓冲液重悬混合均匀咪唑洗脱后的Ni beads

Fig. 6 Analysis of purified His-SUMO-MLL3SET* M：Protein marker. 1：Induced whole cell. 2：Supernatant of the induced cells. 3：Precipitation of the induced cells. 4：Flow through before washing. 5：Flow through after washing with 50 mL buffer. 6：Flow through after washing with 1 L buffer. 7：Suspended Ni beads with buffer before imidazole elution. 8：Imidazole eluted sample. 9：Suspended Ni beads with buffer after imidazole elution

图7 凝胶过滤色谱分离His-SUMO-MLL3SET*

Fig. 7 Separation of His-SUMO-MLL3SET* by gel filtration chromatography

图8 IPCHCGAVNCR肽段二级质谱图

Fig. 8 MS/MS diagram of the IPCHCGAVNCR peptide

图9 多酶级联反应测His-SUMO-MLL3SET*活性 A：M*AR与MAR活性比较;B：SAH浓度与A515的标准曲线

Fig. 9 Activity of His-SUMO-MLL3SET* measured by a multi-enzyme coupling reaction A：Comparison of the activity of M*AR and MAR. B：Standard curve of SAH concentration and A515

参考文献 24

[1]	Shilatifard A. The COMPASS family of histone H3K4 methylases:mechanisms of regulation in development and disease pathogenesis[J]. Annu Rev Biochem, 2012, 81:65-95. doi: 10.1146/annurev-biochem-051710-134100 pmid: 22663077
[2]	Ayton PM, Cleary ML. Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins[J]. Oncogene, 2001, 20(40):5695-5707. pmid: 11607819
[3]	黎彦璟, 陈勇. 组蛋白甲基转移酶MLL1的结构与功能研究进展[J]. 中国细胞生物学学报, 2014, 36(7):857-868.
	Li YJ, Chen Y. Progress in structural and functional studies of histone methyltransferase MLL1[J]. Chin J Cell Biol, 2014, 36(7):857-868.
[4]	Dehé PM, Dichtl B, Schaft D, et al. Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation[J]. J Biol Chem, 2006, 281(46):35404-35412. doi: 10.1074/jbc.M603099200 URL
[5]	Avdic V, Zhang P, Lanouette S, et al. Structural and biochemical insights into MLL1 core complex assembly[J]. Structure, 2011, 19(1):101-108. doi: 10.1016/j.str.2010.09.022 URL
[6]	Zhang Y, Mittal A, Reid J, et al. Evolving catalytic properties of the MLL family SET domain[J]. Structure, 2015, 23(10):1921-1933. doi: S0969-2126(15)00324-X pmid: 26320581
[7]	Southall SM, Wong PS, Odho Z, et al. Structural basis for the requirement of additional factors for MLL1 SET domain activity and recognition of epigenetic marks[J]. Mol Cell, 2009, 33(2):181-191. doi: 10.1016/j.molcel.2008.12.029 pmid: 19187761
[8]	Li Y, Han J, Zhang Y, et al. Structural basis for activity regulation of MLL family methyltransferases[J]. Nature, 2016, 530(7591):447-452. doi: 10.1038/nature16952 URL
[9]	Sasmal DK, Pulido LE, Kasal S, et al. Single-molecule fluorescence resonance energy transfer in molecular biology[J]. Nanoscale, 2016, 8(48):19928-19944. doi: 10.1039/C6NR06794H URL
[10]	Roy R, Hohng S, Ha T. A practical guide to single-molecule FRET[J]. Nat Methods, 2008, 5(6):507-516.
[11]	Zhang J, Fu Y, Lakowicz JR. Enhanced förster resonance energy transfer(FRET)on single metal particle[J]. J Phys Chem C Nanomater Interfaces, 2007, 111(1):50-56. doi: 10.1021/jp062665e URL
[12]	Zhang J, Fu Y, Chowdhury MH, et al. Enhanced förster resonance energy transfer on single metal particle. 2. dependence on donor-acceptor separation distance, particle size, and distance from metal surface[J]. J Phys Chem C, 2007, 111(32):11784-11792. doi: 10.1021/jp067887r pmid: 19890406
[13]	Lee TC, Kang M, Kim CH, et al. Dual unnatural amino acid incorporation and click-chemistry labeling to enable single-molecule FRET studies of p97 folding[J]. Chembiochem, 2016, 17(11):981-984. doi: 10.1002/cbic.v17.11 URL
[14]	Lang K, Chin JW. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins[J]. Chem Rev, 2014, 114(9):4764-4806. doi: 10.1021/cr400355w URL
[15]	Ryu Y, Schultz PG. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli[J]. Nat Methods, 2006, 3(4):263-265.
[16]	Young TS, Ahmad I, Yin JA, et al. An enhanced system for unnatural amino acid mutagenesis in E. coli[J]. J Mol Biol, 2010, 395(2):361-374. doi: 10.1016/j.jmb.2009.10.030 URL
[17]	Smolskaya S, Andreev Y. Site-specific incorporation of unnatural amino acids into Escherichia coli recombinant protein:methodology development and recent achievement[J]. Biomolecules, 2019, 9(7):255. doi: 10.3390/biom9070255 URL
[18]	Lin X, Yu ACS, Chan TF. Efforts and challenges in engineering the genetic code[J]. Life, 2017, 7(1):12. doi: 10.3390/life7010012 URL
[19]	Young DD, Schultz PG. Playing with the molecules of life[J]. ACS Chem Biol, 2018, 13(4):854-870. doi: 10.1021/acschembio.7b00974 URL
[20]	Dorgan KM, Wooderchak WL, Wynn DP, et al. An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases[J]. Anal Biochem, 2006, 350(2):249-255. doi: 10.1016/j.ab.2006.01.004 URL
[21]	Liu ZP. Histone methylation in heart development and cardiovascular disease[J]. Cardiac and Vascular Biology, 2016, 1:125-146.
[22]	Bale TL, Baram TZ, Brown AS, et al. Early life programming and neurodevelopmental disorders[J]. Biol Psychiatry, 2010, 68(4):314-319. doi: 10.1016/j.biopsych.2010.05.028 URL
[23]	Millan MJ. An epigenetic framework for neurodevelopmental disorders:from pathogenesis to potential therapy[J]. Neuropharmacology, 2013, 68:2-82. doi: 10.1016/j.neuropharm.2012.11.015 pmid: 23246909
[24]	Vissers LELM, Gilissen C, Veltman JA. Genetic studies in intellectual disability and related disorders[J]. Nat Rev Genet, 2016, 17(1):9-18. doi: 10.1038/nrg3999 URL