合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 840-851.DOI: 10.12211/2096-8280.2022-048
• 研究论文 • 上一篇
刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健
收稿日期:
2022-09-06
修回日期:
2022-12-01
出版日期:
2023-08-31
发布日期:
2023-09-14
通讯作者:
李健
作者简介:
基金资助:
Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI
Received:
2022-09-06
Revised:
2022-12-01
Online:
2023-08-31
Published:
2023-09-14
Contact:
Jian LI
摘要:
限制性内切酶在分子生物学研究中是一类重要的工具酶,目前主要由异源生物合成的方式进行表达与生产,由于它们对特定的DNA序列(即酶切位点)具有切割活性,在异源表达时会对宿主产生较高的细胞毒性。而无细胞生物合成体系具有操作快捷、灵活高效、无细胞毒性等优势,因此,本研究利用无细胞蛋白合成(cell-free protein synthesis, CFPS)技术进行限制性内切酶的表达制备。本课题组选择3种限制性内切酶EcoRⅠ、BamHⅠ和BsaⅠ作为研究对象,构建线性DNA为表达模板,无需甲基化酶对宿主的保护,在6 h内即可完成蛋白表达。经亲和色谱与凝胶色谱两步纯化,得到了纯度高(95%左右)、酶活相当(EcoRⅠ 3.7 × 105~3.7 × 106 U/mg,BamHⅠ 8.3 × 102~4.1 × 103 U/mg,BsaⅠ 4.4 × 105 ~ 4.4 × 106 U/mg)的目标蛋白。同时,建立了限制性内切酶的实时酶活检测方法,将有助于限制性内切酶的催化和快速筛选研究。本研究所开发的限制性内切酶无细胞表达制备体系,从基因模板构建到纯化蛋白所需时间短(1~2 d)、蛋白产量高(32.5~130 mg/L无细胞反应)、制备效率高(1.3 × 105 ~ 5.7 × 108 U/L无细胞反应),具有较好的普适性,为限制性内切酶的研发与制备生产提供了新的思路。
中图分类号:
刘晚秋, 季向阳, 许慧玲, 卢屹聪, 李健. 限制性内切酶的无细胞快速制备研究[J]. 合成生物学, 2023, 4(4): 840-851.
Wanqiu LIU, Xiangyang JI, Huiling XU, Yicong LU, Jian LI. Cell-free protein synthesis system enables rapid and efficient biosynthesis of restriction endonucleases[J]. Synthetic Biology Journal, 2023, 4(4): 840-851.
引物名称 | 序列 | 用途 |
---|---|---|
linearPro_F | CCTACAGCGTGAGCATTG | 扩增启动子片段 |
linearPro_R | CATATGGTGATGATGATG | |
linearTer_F | GTCGACCGGCTGCTAACA | 扩增终止子片段 |
linearTer_R | CGGATTCAGTCGTCACTCA | |
SUMOPro_F | GGATCTCGACGCTCTCCCT | 扩增启动子+SUMO标签片段 |
SUMOPro_R | AGGTCCCTGAAACAGGACCTCTAAACCACCAATCTGTTCTCTG | |
SUMO_mut_F | TACGACGGTATTCGTATTCAAGCTGATCAGAC | 去除pSUMO质粒中的EcoRⅠ酶切位点 |
SUMO_mut_R | CAGCTTGAATACGAATACCGTCGTACAAGAATC | |
EcoRⅠ_SUMO_F | TTAGAGGTCCTGTTTCAGGGACCTAGCAACAAAAAACAGAGC | 扩增EcoRⅠ基因片段 |
EcoRⅠ_SUMO_R | CATCATCATCACCATATGAGCAACAAAAAACAGAGC | |
sfGFP_SUMO_F | ATTGGTGGTACCGAGCTCATGAGCAAAGGTGAAGAA | 构建质粒pSUMO-sfGFP |
sfGFP_SUMO_R | GAGTGCGGCCGCAAGCTTTTATTTTTCGAACTGCGG | |
BamHⅠ_F | CATCATCATCACCATATGAAAGTGGAAAAAGA | 扩增BamHⅠ基因片段 |
BamHⅠ_R | TGTTAGCAGCCGGTCGACTTATTTGTTTTCCACTTTATC | |
BsaⅠ_F | TTAGAGGTCCTGTTTCAGGGACCTATGGCAAAAAAGCGGAA | 扩增BsaⅠ基因片段 |
BsaⅠ_R | TGTTAGCAGCCGGTCGACTTAATCCAGATCCGCAAA |
表1 本研究中用到的引物序列
Table 1 Oligonucleotide primers used in this study
引物名称 | 序列 | 用途 |
---|---|---|
linearPro_F | CCTACAGCGTGAGCATTG | 扩增启动子片段 |
linearPro_R | CATATGGTGATGATGATG | |
linearTer_F | GTCGACCGGCTGCTAACA | 扩增终止子片段 |
linearTer_R | CGGATTCAGTCGTCACTCA | |
SUMOPro_F | GGATCTCGACGCTCTCCCT | 扩增启动子+SUMO标签片段 |
SUMOPro_R | AGGTCCCTGAAACAGGACCTCTAAACCACCAATCTGTTCTCTG | |
SUMO_mut_F | TACGACGGTATTCGTATTCAAGCTGATCAGAC | 去除pSUMO质粒中的EcoRⅠ酶切位点 |
SUMO_mut_R | CAGCTTGAATACGAATACCGTCGTACAAGAATC | |
EcoRⅠ_SUMO_F | TTAGAGGTCCTGTTTCAGGGACCTAGCAACAAAAAACAGAGC | 扩增EcoRⅠ基因片段 |
EcoRⅠ_SUMO_R | CATCATCATCACCATATGAGCAACAAAAAACAGAGC | |
sfGFP_SUMO_F | ATTGGTGGTACCGAGCTCATGAGCAAAGGTGAAGAA | 构建质粒pSUMO-sfGFP |
sfGFP_SUMO_R | GAGTGCGGCCGCAAGCTTTTATTTTTCGAACTGCGG | |
BamHⅠ_F | CATCATCATCACCATATGAAAGTGGAAAAAGA | 扩增BamHⅠ基因片段 |
BamHⅠ_R | TGTTAGCAGCCGGTCGACTTATTTGTTTTCCACTTTATC | |
BsaⅠ_F | TTAGAGGTCCTGTTTCAGGGACCTATGGCAAAAAAGCGGAA | 扩增BsaⅠ基因片段 |
BsaⅠ_R | TGTTAGCAGCCGGTCGACTTAATCCAGATCCGCAAA |
图2 限制性内切酶EcoRⅠ的无细胞制备(Western blot和SDS-PAGE检测无细胞表达、纯化的限制性内切酶EcoRⅠ)CE—cell extract,以BL21 Star (DE3)制备的细胞提取物;CE+ —含有分子伴侣(pG-KJE8)的BL21 Star(DE3)细胞提取物;T—全菌蛋白;S—可溶蛋白;M—蛋白标准样品;FT—纯化上样穿出液;W—洗杂流出液;Eluate—洗脱液
Fig. 2 Cell-free production of restriction endonuclease EcoRⅠ(Western blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonuclease EcoRⅠ) CE—cell extract of BL21 Star (DE3); CE+ —cell extract of BL21 Star (DE3) with chaperone (pG-KJE8); T—total protein; S—soluble protein; M—marker; FT—flow throughout sample; W—washed sample; Eluate—eluted sample
图4 无细胞制备限制性内切酶EcoRⅠ的酶活测定NC—阴性对照;PC—阳性对照
Fig. 4 Catalytic activity determination of cell-free produced restriction endonuclease EcoRⅠNC—negative control; PC—positive control
图5 限制性内切酶BamHⅠ和BsaⅠ的无细胞制备(Western blot和SDS-PAGE检测无细胞表达纯化的限制性内切酶BamHⅠ和BsaⅠ)
Fig. 5 Cell-free production of restriction endonucleases BamHⅠ and BsaⅠ(Western Blot and SDS-PAGE analyses of cell-free expressed and purified restriction endonucleases BamHⅠ and BsaⅠ)
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