WO2020078321A1 - 一种用于表达Bt杀虫蛋白的高效蛋白质表达系统 - Google Patents

一种用于表达Bt杀虫蛋白的高效蛋白质表达系统 Download PDF

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WO2020078321A1
WO2020078321A1 PCT/CN2019/111040 CN2019111040W WO2020078321A1 WO 2020078321 A1 WO2020078321 A1 WO 2020078321A1 CN 2019111040 W CN2019111040 W CN 2019111040W WO 2020078321 A1 WO2020078321 A1 WO 2020078321A1
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郑茜
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Syngenta Participations AG
Syngenta Biotechnology China Co Ltd
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Syngenta Biotechnology China Co Ltd
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  • the present invention relates to the field of protein (particularly, Bt protein) expression.
  • the invention provides an efficient protein expression system for expressing Bt insecticidal protein.
  • the protein expression system uses Bla ( ⁇ -lactamase) as a fusion tag, thereby efficiently expressing Bt genes that cannot be expressed as soluble proteins into soluble proteins.
  • E. coli In vitro protein expression technology is an important part of genetic engineering technology. Compared with other protein expression systems, E. coli is an efficient and economical approach, and the recombinant protein expression can reach 50% of the total protein quality of E. coli. Recombinant proteins with normal biochemical activity are usually in soluble form, so in order to obtain active proteins, soluble expression pathways are usually used.
  • Bt protein is a parasporal crystal protein formed by Bacillus thuringiensis during the spore formation period. Bt protein is a widely used biological pesticide. It is safe, efficient, and has a broad spectrum of insecticidal activity. Discovering new Bt protein has high commercial application value.
  • Lucigen Solubility & Expression Screening System is a protein expression tag screening kit containing 7 fusion tags. Gene fragments can be easily connected to 8 different vectors through homologous recombination reaction. Through the rapid screening of 7 different fusion tags, the tags most suitable for the expression of the target protein are obtained (see Table 1 below).
  • Bt gene needs to be expressed as a soluble protein to test its insecticidal activity.
  • E. coli expression technology only about 50-60% of Bt genes can be successfully expressed. Therefore, the successful expression of Bt gene as a soluble protein has important research and commercial value.
  • the invention establishes an efficient protein expression system. Using existing E. coli expression technology, about 50-60% of Bt genes can be successfully expressed as soluble proteins. Using the method of the present invention, the remaining 40-50% of the Bt gene that cannot be expressed as soluble protein can be successfully expressed by 80% (expressed as soluble protein), and the total Bt gene expression success rate can reach more than 80%.
  • the invention relates to the use of Bla, ⁇ -lactamase, as a fusion tag for the expression of Bt protein.
  • the present invention relates to the use of the engineered pSol-Bla vector as shown in FIG. 1 for expressing Bt protein.
  • the Bt protein is expressed in E. coli. More preferably, the E. coli is E. coli 10G or BL21 (DE3). Preferably, in the above use, the Bt protein is a Bt protein that cannot be expressed.
  • the invention relates to a fusion protein comprising Bt protein and ⁇ -lactamase as a fusion tag.
  • the Bt protein is a Bt protein that cannot be expressed.
  • the invention relates to a nucleic acid molecule that encodes the aforementioned fusion protein.
  • the invention relates to a vector comprising the above-mentioned nucleic acid molecule.
  • the vector is a pSol-Bla vector inserted with the Bt gene to be expressed, or a modified pSol-Bla vector as shown in FIG. 1 inserted with the Bt gene to be expressed.
  • the present invention relates to a cell comprising the above-mentioned vector.
  • the cell is an E. coli cell. More preferably, the E. coli cells are E. coli 10G or BL21 (DE3) cells.
  • the present invention relates to a method for expressing Bt protein, comprising: (1) culturing the above-mentioned cells under conditions suitable for expressing a desired protein; and (2) optionally, removing the Bla tag To obtain Bt protein without tag.
  • the present invention relates to a modified pSol-Bla vector as shown in FIG.
  • Figure 1 is a map of the modified pSol-Bla vector of the present invention.
  • Example 1 Screening fusion tags for 16 Bt genes that cannot be expressed as soluble proteins to achieve soluble expression
  • Lucigen's Solubility and Expression Screening System kit according to the instructions to design the primer sequences at both ends to synthesize PCR primers for these 16 genes.
  • the target fragments of 16 genes with homologous recombination ends were obtained by PCR amplification.
  • the plasmids are transformed into the chemically competent cells.
  • the obtained monoclonal plasmids identify positive clones by PCR, they are sent to the company for sequencing and identification. After analyzing the sequencing results, we obtained the plasmids of these 16 genes on 8 vectors, a total of 128 plasmids.
  • E. coli 10G or BL21 (DE3) glycerol After cultivating 30 ⁇ l of E. coli 10G or BL21 (DE3) glycerol with these 16 genes in 3 ml of LB medium at 37 ° C to an OD600 of approximately 0.5, add 20% rhamnose to induce culture at 18 ° C overnight. Collect the bacterial cells, collect the whole bacteria and supernatant separately after ultrasonication, and boil them for 10 minutes, then analyze the expression on SDS-PAGE. The expression of these 128 plasmids in E. coli 10G and BL21 (DE3) cells is shown in Table 3 below (where the first 6 genes in Table 3 were purified after successful expression and the Bla tag was excised to obtain Pure soluble protein of interest).
  • Lucigen's pSol-Bla vector the stop codon TAA at positions 1326-1328 in the original vector was removed, and the sequence "ggatccggctcaggctcaggctcaggctcaggctcaggctcactcgag" was added at position 1326 of the original vector, which contains One BamHI site and one XhoI site.
  • the modified pSol-Bla vector is shown in Figure 1.

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Abstract

一种用于表达Bt杀虫蛋白的蛋白质表达系统。所述蛋白质表达系统采用Bla(β-内酰胺酶)作为融合标签，从而将不能表达成可溶性蛋白质的Bt基因表达为可溶性蛋白质。

Description

一种用于表达Bt杀虫蛋白的高效蛋白质表达系统 技术领域
本发明涉及蛋白质(特别地，Bt蛋白)表达的领域。本发明提供了一种用于表达Bt杀虫蛋白的高效蛋白质表达系统。所述蛋白质表达系统采用Bla(β-内酰胺酶)作为融合标签，从而高效地将不能表达成可溶性蛋白质的Bt基因表达为可溶性蛋白质。
背景技术
蛋白质的体外表达技术是基因工程技术中的一个重要部分。大肠杆菌相比其他蛋白质表达系统来说是一种高效、经济的途径，重组蛋白表达量可达到大肠杆菌总蛋白质量的50％。具有正常生化活性的重组蛋白通常为可溶性形式，因而为了得到有活性的蛋白质，通常采用可溶性表达途径。
Bt蛋白是苏云金芽孢杆菌(Bacillus thuringiensis)在芽孢形成时期在菌体内形成的伴孢晶体蛋白质。Bt蛋白是一种广泛应用的生物农药，安全且具有高效、广谱的杀虫活性，且对人畜及非靶标害虫无害。发掘新的Bt蛋白具有很高的商业应用价值。
Lucigen公司的
Figure PCTCN2019111040-appb-000001
Solubility&Expression Screening System是一个含有7种融合标签的蛋白质表达标签筛选试剂盒。基因片段通过同源重组反应，可以很方便地连接到8个不同的载体上。通过7种不同融合标签的快速筛选，从而得到最适合目的蛋白质表达的标签(见下面的表1)。
表1
Figure PCTCN2019111040-appb-000002
新发掘的Bt基因需要表达成为可溶性的蛋白质才能测试其杀虫活性。但是依靠目前的大肠杆菌表达技术，只有约50～60％的Bt基因能够被顺利表达。因此，将Bt基因成功表达为可溶性的蛋白质具有重要的研究和商业价值。
发明内容
本发明建立了一种高效的蛋白质表达系统。使用现有的大肠杆菌表达技术，约有50～60％的Bt基因能够被成功表达为可溶性的蛋白质。使用本发明的方法可以将剩下不能表达成可溶性蛋白质的40～50％的Bt基因实现80％的成功表达(表达为可溶性蛋白质)，使总的Bt基因表达成功率达到80％以上。
本发明最初以16个不能表达成可溶性蛋白质的Bt基因做为测试样本，采用Lucigen公司的
Figure PCTCN2019111040-appb-000003
Solubility&Expression Screening System中的8个载体对这16个基因进行融合标签的筛选，结果显示pSol-Bla载体能够对16个测试样品实现100％的表达。为了方便克隆，我们对Lucigen的pSol-Bla载体进行了一些改造，并用改造后的载体 对另外的237个不能表达成可溶性蛋白质的Bt基因在大肠杆菌中进行了表达；结果有193个基因成功表达成为可溶性蛋白质，表达成功率为81.4％。因此，对于总共253(16+237)个之前不能表达成可溶性蛋白质的Bt基因，结果有209(16+193)个基因成功表达成为可溶性蛋白质，表达成功率为82.6％。
在一个方面，本发明涉及Bla即β-内酰胺酶作为融合标签用于表达Bt蛋白的用途。
在另一个方面，本发明涉及如图1所示的经改造的pSol-Bla载体用于表达Bt蛋白的用途。
优选地，在上述用途中，在大肠杆菌中表达Bt蛋白。更优选地，所述大肠杆菌为大肠杆菌10G或者BL21(DE3)。优选地，在上述用途中，所述Bt蛋白为不能表达的Bt蛋白。
在另一个方面，本发明涉及一种融合蛋白，其包含Bt蛋白和作为融合标签的β-内酰胺酶。优选地，所述Bt蛋白为不能表达的Bt蛋白。
在另一个方面，本发明涉及一种核酸分子，其编码上述的融合蛋白。
在另一个方面，本发明涉及一种载体，其包含上述的核酸分子。优选地，所述载体为插入有待表达的Bt基因的pSol-Bla载体，或者插入有待表达的Bt基因的如图1所示的经改造的pSol-Bla载体。
在另一个方面，本发明涉及一种细胞，其包含上述的载体。优选地，所述细胞为大肠杆菌细胞。更优选地，所述大肠杆菌细胞为大肠杆菌10G或者BL21(DE3)细胞。
在另一个方面，本发明涉及用于表达Bt蛋白的方法，其包括：(1)在适合于表达出所需的蛋白质的条件下培养上述的细胞；和(2)任选地，切除Bla标签，从而获得不含标签的Bt蛋白。
在另一个方面，本发明涉及如图1所示的经改造的pSol-Bla载体。
附图说明
图1为本发明的经改造的pSol-Bla载体的图谱。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1.为16个不能表达成可溶性蛋白质的Bt基因筛选融合标签以实现可溶性表达
我们选择了16个用常规方法(例如，pET系列载体在不同温度和IPTG诱导浓度下的表达，使用SUMO，GST等融合标签，使用不同种类的感受态细胞，无细胞培养，用Bt细胞表达，等等)无法表达成可溶性蛋白质的Bt基因，它们的编号和分子量大小以及表达的问题如下面的表2所示：
表2
蛋白质编号 基因家族 SEQ ID NO 分子量 表达的问题
BT-0025 Cry1If 1 80KD 不可溶
BT-0027   2 79KD 无表达
BT-0275 Cry22Aa 3 96KD 无表达
BT-0362   4 73KD 无表达
BT-0425 Cry36Aa 5 56KD 无表达
BT-0435 Cry42Aa 6 75KD 无表达
BT-0445 Vip1Aa 7 138KD 不可溶
BT-0452 Cry19Ca 8 59KD 无表达
BT-0560 Cry20Aa 9 26KD 无表达
BT-0587 Cry49Ab 10 60KD 无表达
BT-0669 Cry2Ae 11 30KD 不可溶
BT-0685 Cry49Ab 12 51KD 不可溶
BT-0827 Cry48Ab2 13 86KD 无表达
BT-0830 Cry48Ab2 14 87KD 无表达
BT-0831 Cry54Ba1 15 80KD 无表达
HO4 Cry1Ab.1Ca 16 130KD 不可溶
使用Lucigen公司的
Figure PCTCN2019111040-appb-000004
Solubility and Expression Screening System试剂盒，按照其说明书设计两端的引物序列分别合成这16个基因的PCR引物。通过PCR扩增的方法分别获得带有同源重组末端的16个基因的目的片段。然后，在将PCR产物与试剂盒中的8个线性载体混合后对化学感受态细胞进行质粒的转化。在将获得的单克隆质粒通过PCR鉴定出阳性克隆后送公司进行测序鉴定。对测序结果分析之后，我们分别获得了这16个基因在8个载体上的质粒，一共128个质粒。
在将30μl的带有这16个基因的大肠杆菌10G或者BL21(DE3)甘油菌在3ml LB培养基中于37℃培养至OD600约为0.5后，加入20％的鼠李糖于18℃诱导培养过夜。收集菌体，超声波破碎后分别收集全菌和上清液，煮沸10分钟后上SDS-PAGE分析表达情况。这128个质粒在大肠杆菌10G和BL21(DE3)细胞中的表达情况如下面的表3所示(其中表3中的前6个基因在成功表达之后进行了纯化并且把Bla标签切除，得到了纯的可溶性目的蛋白)。
表3
Figure PCTCN2019111040-appb-000005
Figure PCTCN2019111040-appb-000006
结果显示，所述试剂盒中的8个带有不同标签的载体大多数能表达这16个Bt基因中的个别基因，只有pSol-Bla载体对这16个基因全部有效(即能够所述基因表达成可溶性蛋白质)，这暗示着Bla标签对于Bt基因的表达有特异性提高的效果。
实施例2.改造Lucigen公司的pSol-Bla载体并用在237个不能表达成可溶性蛋白质的Bt基因上
为了方便克隆，我们将Lucigen公司的pSol-Bla载体做了少许改造：去掉了原载体中位置1326-1328处的终止密码子TAA，并且在原载体的位置1326处添加了序列“ggatccggctcaggctcaggctcaggctcaggctcaggctcactcgag”，其中包含一个 BamHI酶切位点和一个XhoI酶切位点。改造后的pSol-Bla载体如图1所示。我们选择了237个不能表达成可溶性蛋白质的Bt基因(采用pET28a载体，在18℃下用0.2-0.5mM IPTG诱导过夜的条件下无法在细胞裂解液的上清中获得可溶性形式的蛋白质)，用酶切连接方法将他们克隆到改造后的pSol-Bla载体上。转化至大肠杆菌10G细胞之后，我们使用同样的方法表达了这237个质粒：在将30μl甘油菌加至3ml LB培养基中于37℃培养至OD600约为0.5后，加入20％的鼠李糖于18℃诱导培养过夜。收集菌体，超声波破碎后分别收集全菌和上清液，煮沸10分钟后上SDS-PAGE分析表达情况。
结果显示，237个不能表达成可溶性蛋白质的Bt基因中有193个通过这种方式获得了可溶性的融合蛋白，成功率高达81.4％。这个结果表明，融合有Bla的载体可以特异性地提升Bt基因的成功表达成功率。
所述237个不能表达成可溶性蛋白质的Bt基因的信息如下：
表4
Figure PCTCN2019111040-appb-000007
Figure PCTCN2019111040-appb-000008
Figure PCTCN2019111040-appb-000009
Figure PCTCN2019111040-appb-000010
Figure PCTCN2019111040-appb-000011
Figure PCTCN2019111040-appb-000012
Figure PCTCN2019111040-appb-000013
参考文献
1.Tokunaga Hi等人,(2010)Appl Microbiol Biotechnol.88,1223
2.Bruce E.Tabashnik等人,Sci Rep.2015；5:15107

Claims (15)

  1. Bla即β-内酰胺酶作为融合标签用于表达Bt蛋白的用途。
  2. 如图1所示的经改造的pSol-Bla载体用于表达Bt蛋白的用途。
  3. 根据权利要求1或2所述的用途，其中在大肠杆菌中表达Bt蛋白。
  4. 根据权利要求3所述的用途，其中所述大肠杆菌为大肠杆菌10G或者BL21(DE3)。
  5. 根据权利要求1-4中任一项所述的用途，其中所述Bt蛋白为不能表达的Bt蛋白。
  6. 融合蛋白，其包含Bt蛋白和作为融合标签的β-内酰胺酶。
  7. 根据权利要求6所述的融合蛋白，其中所述Bt蛋白为不能表达的Bt蛋白。
  8. 核酸分子，其编码根据权利要求6或7所述的融合蛋白。
  9. 载体，其包含根据权利要求8所述的核酸分子。
  10. 根据权利要求9所述的载体，其中所述载体为插入有待表达的Bt基因的pSol-Bla载体，或者插入有待表达的Bt基因的如图1所示的经改造的pSol-Bla载体。
  11. 细胞，其包含根据权利要求9或10所述的载体。
  12. 根据权利要求11所述的细胞，其中所述细胞为大肠杆菌细胞。
  13. 根据权利要求12所述的细胞，其中所述大肠杆菌细胞为大肠杆菌10G或者BL21(DE3)细胞。
  14. 用于表达Bt蛋白的方法，其包括：
    (1)在适合于表达出所需的蛋白质的条件下培养根据权利要求11-13中任一项所述的细胞；和
    (2)任选地，切除Bla标签，从而获得不含标签的Bt蛋白。
  15. 如图1所示的经改造的pSol-Bla载体。
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