Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (3): 194-202.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0465
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WANG Xiao-qin1(), HUANG Yin-ping1, WANG Wei-qian2, WU Ping2, QUAN Shu1()
Received:
2021-04-09
Online:
2022-03-26
Published:
2022-04-06
Contact:
QUAN Shu
E-mail:wxq870718522@163.com;shuquan@ecust.edu.cn
WANG Xiao-qin, HUANG Yin-ping, WANG Wei-qian, WU Ping, QUAN Shu. Expression and Purification of the MLL3SET Protein with a Site-directed Mutation of an Unnatural Amino Acid[J]. Biotechnology Bulletin, 2022, 38(3): 194-202.
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 |
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 |
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
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
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℃
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
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
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
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