WO2024016429A1 - 一种高酯键形成效率分子肽突变体 - Google Patents

一种高酯键形成效率分子肽突变体 Download PDF

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WO2024016429A1
WO2024016429A1 PCT/CN2022/115683 CN2022115683W WO2024016429A1 WO 2024016429 A1 WO2024016429 A1 WO 2024016429A1 CN 2022115683 W CN2022115683 W CN 2022115683W WO 2024016429 A1 WO2024016429 A1 WO 2024016429A1
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molecular peptide
mutant
recombinant plasmid
protein
bond formation
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江凌
陈耀
陈昱金
朱丽英
刘伟
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南京工业大学
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12R2001/19—Escherichia coli
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of molecular peptide design, and specifically relates to a molecular peptide mutant with high ester bond formation efficiency.
  • the present invention uses rational design to transform Catcher to obtain a mutant EBCatcher, which can quickly form a covalent bond with EBTag.
  • the present invention introduces three mutations into Catcher, which can greatly improve the covalent bond formation efficiency of Tag and Catcher.
  • the modified Catcher is called EBCatcher
  • the Tag is called EBTag.
  • a molecular peptide mutant with high ester bond formation efficiency the amino acid sequence is shown in SEQ ID NO: 1.
  • Another object of the present invention is to provide a gene sequence encoding the molecular peptide mutant of claim 1.
  • Another object of the present invention is to provide the application of the molecular peptide mutant in protein separation.
  • Another object of the present invention is to provide a purification method for the molecular peptide mutant, including:
  • pET-22b is selected as the vector.
  • the enzyme cleavage sites connected to the vector are Nde I and Xho I.
  • the host bacterium is E. coli BL21 (DE3).
  • the E. coli containing the recombinant plasmid is cultured in LB medium.
  • the supernatant is subjected to protein purification in Ni-NTA resin.
  • the purified protein is dialyzed in a 3000Da dialysis bag for 24-26 hours.
  • the modified EBCatcher can still form isopeptide bonds with EBTag, but the covalent bond formation efficiency is significantly increased.
  • Figure 1 is the sequence alignment of Catcher and EBCatcher.
  • Figure 2 shows the connection efficiency of EBCatcher-GFP and EBTag-GFP.
  • Figure 3 shows the connection efficiency of Catcher-GFP and EBTag-GFP.
  • the original protein crystal structure in the example was obtained from the PDB database, and the PDB ID is 4ni6.
  • This example specifically explains the method of designing mutants.
  • This example specifically illustrates the purification method of the mutant.
  • the mutated molecular peptide was fully gene synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and a GFP was added to the EBTag and EBCatcher at the same time to increase the molecular weight.
  • the recombinant plasmid pET-22b-EBCatcher-GFP was obtained by cloning on the vector pET-22b.
  • the enzyme cutting sites are Nde I and Xho I, and the host is Escherichia coli E.coli BL21 (DE3).
  • the recombinant vector of EBTag-GFP is pET-22b-EBTag-GFP, and the restriction site and host are the same as EBCatcher-GFP.
  • This example tests the connection efficiency of EBCatcher-GFP and Catcher-GFP.
  • Experimental group Mix EBTag-GFP and EBCatcher-GFP in Example 1 in 0.1M pH 6.0 phosphate buffer (containing 20% glycerol and 100 ⁇ M CaCl 2 ) at a concentration of 1:1 at 10 ⁇ M, and incubate at 20°C. The reaction was carried out for 60 minutes, during which samples were taken every 10 minutes, and SDS-PAGE was used to measure the ligation efficiency.
  • Control group Complete gene synthesis of the Catcher-GFP gene was performed at Sangon Bioengineering (Shanghai) Co., Ltd., and the other methods were the same as in Example 2 and the experimental group.
  • BufferA is a pH 8.0 0.1M phosphate buffer, dissolved in 500mM NaCl and 20mM imidazole;
  • BufferB is a pH 8.0 0.1M phosphate buffer, dissolved in 500mM NaCl and 300mM imidazole;
  • Ni-NTA prepacked column was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. The remaining reagents are commercially available.
  • SDS-PAGE protein gel kit to configure a 12% separating gel and a 5% stacking gel. Take 10 ⁇ L of the prepared sample and load it, the voltage is 120V, and the electrophoresis time is 120min. After completion of the staining, use Coomassie Brilliant Blue staining solution for 60-120 minutes, and then use destaining solution to destain until the background is transparent. Take a picture with a gel imager and use ImageJ to perform band density analysis to get the connection efficiency.
  • the SDS-PAGE protein gel kit was purchased from Beijing Solebao Technology Co., Ltd., and the other reagents were commercially available.

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Abstract

提供一种高酯键形成效率分子肽突变体，其氨基酸序列如 SEQ ID NO:1所示。在基本不影响 Catcher空间结构的基础上引入了三个位点的突变，通过三种联合突变得到突变体 EBCatcher， EBCatcher 极大程度地提高了 Catcher 在水溶液中的稳定性，可以提高与EBTag的结合效率。

Description

一种高酯键形成效率分子肽突变体 技术领域
本发明属于分子肽设计领域，具体涉及一种高酯键形成效率分子肽突变体。
背景技术
在2014年，Edward N.Baker等人发现了Thr-Gln之间形成异肽键Ig样蛋白，并进行了结构解析，其晶体结构的PDB ID为4ni6；该团队于2017年在4ni6的基础上，将其分离为Catcher和Tag。在偏酸性环境下，Catcher的Thr11和Tag的Gln14可以形成共价键(酯键)，但是需要在体系中加入甘油和CaCl 2，与普通的分子肽对不同的是，将环境的pH调成8.0，并将甘油和Ca 2+透析出去后，酯键可以被水解。这将被应用在蛋白分离等领域。但是其共价键形成效率不高，影响了其拓展应用。
发明内容
本发明利用理性设计，将Catcher改造得到突变体EBCatcher，可以快速与EBTag形成共价键。
本发明在Catcher上引入3个突变，可以大幅度提高Tag和Catcher的共价键形成效率。本发明将改造得到的Catcher称之为EBCatcher，Tag称为EBTag。
一种高酯键形成效率分子肽突变体，氨基酸序列如SEQ ID NO：1所示。
本发明的另一目的在于提供编码权利要求1所述分子肽突变体的基因序列。
本发明的又一目的在于提供所述分子肽突变体在蛋白质分离中的应用。
本发明的再一目的在于提供所述分子肽突变体的纯化方法，包括：
(1)将分子肽的基因序列导入载体构建重组质粒，重组质粒导入宿主菌；
(2)将含重组质粒的宿主菌培养至OD 600＝0.6-0.8，之后加入IPTG诱导；
(3)诱导结束后，取菌液离心后收集菌体，加入磷酸缓冲液重悬，超声破碎；
(4)破碎液超速离心后取上清液，纯化透析获取纯化蛋白。
作为一种优选的实施方式，所述(1)中，载体选用pET-22b。
作为一种优选的实施方式，载体连接的酶切位点为Nde I和Xho I。
作为一种优选的实施方式，所述(1)中，宿主菌为大肠杆菌E.coli BL21(DE3)。
作为一种优选的实施方式，所述(2)中，含重组质粒的大肠杆菌在LB培养基中培养。
作为一种优选的实施方式，所述(4)中，上清液在Ni-NTA树脂中进行蛋白纯化。
作为一种优选的实施方式，纯化后的蛋白在3000Da的透析袋中透析24-26h。
改造后的EBCatcher仍然可以与EBTag形成异肽键，但是共价键形成效率显著增加。
附图说明
图1是Catcher和EBCatcher序列比对。
图2是EBCatcher-GFP和EBTag-GFP连接效率。
图3是Catcher-GFP和EBTag-GFP连接效率。
具体实施方式
实施例中原始的蛋白晶体结构从PDB数据库中获得，PDB ID为4ni6。
实施例1
本实施例具体说明突变体的设计方法。
通过观察原始的蛋白晶体结构对Catcher的氨基酸序列进行突变设计。F31所处位置为钙离子结合位点，在Phe上引入一个羟基变成Tyr(F31Y)，提高了局部的亲水性，并且不改变空间结构，降低在水溶液中的自由能，提高稳定性；而F94所处Catcher的内部，将Phe突变成侧链体积更小的Ile，可以有效降低内部的空间位阻，提高折叠的稳定性；在Q97位引入一个极性氨基酸Glu，在基本不影响结构的前提下，降低在水溶液中的折叠能量。通过三种联合突变，可以极大程度地提高EBCatcher在水溶液中的稳定性，可以提高与EBTag的结合效率。
实施例2
本实施例具体说明突变体的纯化方法。
将突变后的分子肽在生工生物工程(上海)股份有限公司进行全基因合成，将EBTag和EBCatcher上同时加入一个GFP以增加分子量。克隆在载体pET-22b上得到重组质粒pET-22b-EBCatcher-GFP，酶切位点为Nde I和Xho I，宿主为大肠杆菌E.coli BL21(DE3)。EBTag-GFP的重组载体为pET-22b-EBTag-GFP，酶切位点和宿主与EBCatcher-GFP相同。
将带有重组质粒的E.coli BL21(DE3)在LB培养基中37℃中培养至OD 600＝0.6，加入1M的IPTG至终浓度0.5mM，20℃诱导12h。
诱导结束后，12000rpm离心菌液收集菌体，并加入4mL磷酸缓冲液，震荡重悬，在350W的功率下超声破碎15min。
将破碎液在12000rpm，4℃的环境中超速离心20min，取上清液，在Ni-NTA树脂中进行蛋白纯化，纯化后的蛋白在14000Da的透析袋中透析26h后备用。
实施例3
本实施例和实施例1的不同之处仅在于：
将带有重组质粒的E.coli BL21(DE3)在LB培养基中37℃中培养至OD 600＝0.8，加入1M的IPTG至终浓度0.5mM，25℃诱导12h。
实施例4
本实施例测试了EBCatcher-GFP和Catcher-GFP的连接效率。
实验组：将实施例1中的EBTag-GFP和EBCatcher-GFP按照10μM浓度1:1的浓度在0.1MpH 6.0磷酸缓冲液缓(含20％甘油和100μM的CaCl 2)中混合，在20℃环境中反应60min，期间每隔10min取样，利用SDS-PAGE测得连接效率。
对照组：在生工生物工程(上海)股份有限公司进行全基因合成Catcher-GFP基因，其余方法都与实施例2和实验组相同。
其中蛋白纯化的方法：
将1mL的Ni-NTA预装柱中20％的乙醇保护液流尽，并且加入3-4倍柱体积的BufferA来替换填料中的乙醇。将超速离心后的蛋白样品倒入中填料中流尽。再加入3-4柱体积的BufferA进行洗脱，去除吸附在填料上的杂蛋白。再加入3-4倍柱体积的BufferB，洗脱目的蛋白。
BufferA为pH8.0 0.1M的磷酸缓冲液，溶解有500mM的NaCl和20mM的咪唑；
BufferB为pH8.0 0.1M的磷酸缓冲液，溶解有500mM的NaCl和300mM的咪唑；
1mL Ni-NTA预装柱购自生工生物工程(上海)股份有限公司。其余试剂均为市售。
SDS-PAGE蛋白凝胶电泳方法：
将30μL的样品和10μL的4×loading buffer混合，在100℃的金属浴中保温10min，保温结束后降温至4℃后1000-12000rpm离心。利用SDS-PAGE蛋白凝胶试剂盒配置12％的分离胶和5％的浓缩胶。将制备的样品取10μL上样，电压为120V，电泳时间为120min。结束后使用考马斯亮蓝染色液染色60-120min后，利用脱色液脱色至背景透明。在凝胶成像仪拍照，利用ImageJ进行条带密度分析，即可得到连接效率。其中SDS-PAGE蛋白凝胶试剂盒购自北京索莱宝科技有限公司，其余试剂均为市售。
结果分别如图2和图3所示(纵坐标为蛋白复合物的产量)，可以看出，由本发明计算得到的分子肽突变体EBCatcher不仅依旧具备与EBTag形成异肽键的能力，并且共价键形成速率显著提高。此外，实施例2中蛋白诱导表达结果和实施例1有轻微差别，但对最终共价键形成速率没有明显影响。

Claims (10)

  1. 一种高酯键形成效率分子肽突变体，其特征在于，氨基酸序列如SEQ ID NO：1所示。
  2. 编码权利要求1所述分子肽突变体的基因序列。
  3. 权利要求1所述分子肽突变体在蛋白质分离中的应用。
  4. 权利要求1所述分子肽突变体的纯化方法，其特征在于，包括：
    (1)将分子肽的基因序列导入载体构建重组质粒，重组质粒导入宿主菌；
    (2)将含重组质粒的宿主菌培养至OD 600＝0.6-0.8，之后加入IPTG诱导；
    (3)诱导结束后，取菌液离心后收集菌体，加入磷酸缓冲液重悬，超声破碎；
    (4)破碎液超速离心后取上清液，纯化透析获取纯化蛋白。
  5. 根据权利要求4所述的方法，其特征在于，所述(1)中，载体选用pET-22b。
  6. 根据权利要求4所述的方法，其特征在于，载体连接的酶切位点为Nde I和Xho I。
  7. 根据权利要求4所述的方法，其特征在于，所述(1)中，宿主菌为大肠杆菌E.coli BL21(DE3)。
  8. 根据权利要求4所述的方法，其特征在于，所述(2)中，含重组质粒的大肠杆菌在LB培养基中培养。
  9. 根据权利要求4所述的方法，其特征在于，所述(4)中，上清液在Ni-NTA树脂中进行蛋白纯化。
  10. 根据权利要求4所述的方法，其特征在于，纯化后的蛋白在3000 Da的透析袋中透析24-26h。
PCT/CN2022/115683 2022-07-22 2022-08-30 一种高酯键形成效率分子肽突变体 WO2024016429A1 (zh)

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