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Expression and Purification of the Eukaryotic MBP-MOS1 Transposase from Sf21 Insect Cells
Sf21昆虫细胞中真核MBP-MOS1转座酶的表达和纯化   

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Abstract

Here, we present the full-length protocol for purifying the recombinant MOS1 transposase from insect cells used in our recent publication (Pflieger et al., 2014), which involved a N-terminal MBP-tag and maltose-affinity chromatography.
Due to their overall basic properties, transposases are often difficult to purify, especially because they tend to aggregate. Since the 90s, we chose a method of purification without a denaturation step. Our first priority was to preserve the 3D structure of the protein in order to maintain its biochemical activities with the highest specific activity. Nevertheless, our production/purification made from bacteria regularly contain truncated products (or degradation products) and their levels increase with concentration of purified transposase. In contrast, production/purification made from eukaryotic cells do not contain such degradation product. We thus developed a protocol involving the pVL1392 baculovirus transfer vector and the BaculoGoldTM baculovirus expression system, allowing the expression of recombinant MOS1 from baculovirus-infected Sf21 cells.

Materials and Reagents

  1. Sf21 cells (Life Technologies, Gibco®, catalog number: 11497 )
  2. Insect Express Sf9/S2 medium (PAA, catalog number: E15-875 )
  3. Fetal bovine serum (Life Technologies, Gibco®)
  4. Baculogold baculovirus expression system (BD Biosciences, catalog numbers: 554739 and 560129 )
  5. Anti-MBP anti-body (Santa Cruz, catalog number: sc-808 )
  6. Tris.HCl (pH 7.6)
  7. NaCl
  8. DTT
  9. NP40
  10. Antiprotease cocktails (like Roche’s EDTA-free cocktail tablets, catalog number: 11-873 580-001 )
  11. Maltose
  12. Bovine serum albumin (BSA)
  13. Lysis buffer (see Recipes)
  14. Binding buffer (see Recipes)
  15. Elution buffer (see Recipes)

Equipment

  1. 25 cm2, 150 cm2 flask (usual culture-cell equipment)
  2. 1 ml prepacked column (GE Healthcare, MBPTrap™ HP) or 1 ml of amylose resin in batch (New England Biolabs)

Procedure

  1. Sf21 culture conditions
    1. Sf21 cells are cultivated in Insect Express Sf9/S2 medium (PAA) containing 5% fetal bovine serum (or FBS, PAA) at 27 °C (herein called “complete media”).
    2. Every 4 to 6 days, a new 25 cm2 flask is inoculated with 1 to 1.25 x 106 viable cells in 5 ml of complete medium.

  2. Generating a recombinant baculovirus that expresses the MBP-MOS1 fusion protein in Sf21 cells
    1. The MBP-MOS1 coding sequence is cloned into the pVL-1392 at the SmaI restriction site.
    2. High-titre, low-passage recombinant Baculovirus stocks are obtained in Sf21 cells following the instructions of the “Baculogold baculovirus expression system” manufacturer (BD Biosciences) (https://www.bdbiosciences.com/documents/Baculovirus_vector_system_manual.pdf).
    3. 5 to 10 batches of recombinant Baculoviruses are assayed for their ability to produce the recombinant MBP-MOS1 in infected Sf21 cells by Western blot (https://www.sciencepub.net/researcher/0102/09_1259_yang_west_research0102.pdf) with the anti-MBP anti-body (dilution 1/10,000). The most efficient isolate is selected for further protein production processes.

  3. Producing the MBP-MOS1 fusion protein in Sf21 cells
    1. The selected baculovirus stock is used to infect Sf21 cells.
    2. Twenty-one 150 cm2 flasks with 6 x 107 viable cells per flask in 30 ml of complete medium are prepared and incubated at 27 °C until the cells reach confluence (see Figure 1).


      Figure 1. Confluent non-infected Sf21 cells

    3. The medium is then replaced by 10 ml of medium without FBS and cells are infected with 100 µl of high titre recombinant Baculoviruses stock solution (virus titer should be 1 to 2 x 108 pfu/ml).
    4. Cells are incubated for 3 h at 27 °C and then 5 ml of complete medium (with 5% FBS) is added. The cells are incubated for 3 days at 27 °C, in the dark.
    5. Check for signs of infection 2-3 days after inoculation. Cells should be enlarged in size (about 2 fold) and a large nucleus should be visible (see Figure 2).


      Figure 2. Infected Sf21 cells

    6. To pellet cells, gently dislodge cells from monolayers by smacking and transfer the cell suspension to a sterile centrifuge tube of appropriate size. Spin down cells at 10,000 x g for 5 min. The MBP-MOS1 protein will be found in the cell pellet, which can be stored at -80 °C or immediately processed.

  4. MBP-MOS1 fusion protein extraction in crude extracts
    1. Pellets are washed in 1x PBS and pooled so that one pellet contains the cells coming from three 150 cm2 flasks.
    2. Cytoplasmic crude extract (CCE).
      1. Each pellet is resuspended in 3.5 ml of lysis buffer.
      2. Cells are lysed on ice for 30 min and then centrifuged at 15,000 x g and 4 °C for 30 min. The supernatant (≈30 ml) corresponds to the cytoplasmic crude extract (CCE).
      3. Protein concentration is usually around 5 to 10 µg/µl (i.e. a total amount of about 125 to 250 mg), as determined by Bradford assay (http://web.mnstate.edu/provost/bradfordproteinassayprotocol.pdf) using BSA as a standard. Keep the CCE on ice.
    3. Nuclear crude extract (NCE)
      1. Each pellet is resuspended in 0.75 ml of lysis buffer without NP40.
      2. Sonicated on ice (Ultrasonic Processor, output power at 100%, 3 pulses of 15 sec each) and centrifuged at 10,000 x g and 4 °C for 10 min. The supernatant (≈ 6 ml) corresponds to the nuclear crude extract (NCE).
      3. Protein concentration is usually around 5 to 10 µg/µl (i.e. a total amount of about 30 to 50 mg). Keep the NCE on ice.

  5. MBP-MOS1 fusion protein purification
    CCE and NCE previously obtained can be processed either separately or pooled. In the following, CCE and NCE are pooled together, giving a whole cellular crude extract. MBP-MOS1 can be purified by two ways: Using a 1 ml prepacked column or 1 ml of amylose resin in batch.
    1. Prepacked column
      1. Equilibrate the column with at least 5 ml of binding buffer at 0.5 ml/min.
      2. Adjust the crude extract concentration to 1 mg/ml with the NCE lysis buffer. Apply the sample.
      3. Wash with at least 10 ml of binding buffer (until no material appears in the flow-through). Elute with 5 ml of elution buffer, and collect into 0.5 ml fractions.
      4. After one column volume (about 1 ml), the peak of purified MBP-MOS1 is eluted within two to three fractions, corresponding to those having the maximum UV absorbance at 280 nm.
      5. Pool the positive fractions and store as 50 µl aliquots at -80 °C.
    2. Amylose resin in batch
      1. Wash the resin with at least 5 ml of binding buffer. Add 1 ml of resin to the crude extract.
      2. Incubate for 1 h at 4 °C with gentle mixing. Centrifuge at 800 x g and 4 °C for 5 min. Discard the supernatant.
      3. Wash the resin pellet with at least 10 ml of binding buffer. Centrifuge at 800 x g and 4 °C for 5 min. Discard the supernatant. This is to be repeated five times.
      4. Elute the MPB-MOS1 with 1 ml of elution buffer; Centrifuge at 800 x g and 4 °C for 5 min. Recover the supernatant (E1 fraction).
      5. Elution can be repeated twice, giving E2 and E3 fractions.
      6. Highly concentrated fractions (E1 and often E2) are stored as 50 µl aliquots at -80 °C.
      7. MBP-MOS1 concentration in positive fractions (from both methods) is determined by Bradford assay using BSA as a standard.
      8. From twenty F1 50 cm2 infected flasks, 1-2 ml at about 0.5 to 1 µg/µl is expected, depending on the efficiency of the infection.

Recipes

  1. Lysis buffer
    20 mM Tris.HCl (pH 7.6)
    100 mM NaCl
    1 mM DTT
    1% NP40
    Antiprotease cocktails (according to the manufacturer’s recommendations)
  2. Binding buffer
    20 mM Tris.HCl (pH 9)
    100 mM NaCl
    1 mM DTT
  3. Elution buffer
    Binding buffer containing 10 mM maltose

Acknowledgments

Dussassois-Montagne was a doctoral fellow of the French government (MRT). This work was supported by the Région Centre (InhDDE project), the French ANR (Elegineer project), the University of Tours, and the Centre National de la Recherche Scientifique.

References

  1. Pflieger, A., Jaillet, J., Petit, A., Auge-Gouillou, C. and Renault, S. (2014). Target capture during Mos1 transposition. J Biol Chem 289(1): 100-111.

简介

在这里,我们提出了用于从我们最近出版物(Pflieger等,2014)中使用的昆虫细胞中纯化重组MOS1转座酶的全长方案,其涉及N-末端MBP标签和麦芽糖亲和层析。
由于其总体基本性质,转座酶通常难以纯化,特别是因为它们倾向于聚集。自九十年代以来,我们选择了一种纯化方法,而没有变性步骤。我们的首要任务是保留蛋白质的3D结构,以保持其具有最高比活度的生化活性。然而,我们由细菌制成的生产/纯化定期含有截短产物(或降解产物),并且其水平随着纯化转座酶的浓度而增加。相比之下,由真核细胞制成的生产/纯化不含有这样的降解产物。因此,我们开发了涉及pVL1392杆状病毒转移载体和BaculoGoldTM杆状病毒表达系统的方案,从杆状病毒感染的Sf21细胞中表达重组MOS1。

材料和试剂

  1. < em> Sf21细胞(Life Technologies,Gibco ,目录号:11497)。
  2. Insect Express Sf9/S2培养基(PAA,目录号:E15-875)
  3. 胎牛血清(Life Technologies,Gibco )
  4. 杆状病毒杆状病毒表达系统(BD Biosciences,目录号:554739和560129)
  5. 抗MBP抗体(Santa Cruz,目录号:sc-808)
  6. 盐酸(pH7.6)
  7. NaCl
  8. DTT
  9. NP40
  10. 抗蛋白酶鸡尾酒(如Roche的不含EDTA的鸡尾酒片,目录号:11-873 580-001)
  11. 麦芽糖
  12. 牛血清白蛋白(BSA)
  13. 裂解缓冲液(见配方)
  14. 绑定缓冲区(参见配方)
  15. 洗脱缓冲液(见配方)

设备

  1. 25cm 2,150cm 2烧瓶(通常的培养细胞设备)。
  2. 1ml预填充柱(GE Healthcare,MBPTrap TM HP)或1ml批量(New England Biolabs)中的直链淀粉树脂

程序

  1. Sf21 培养条件
    1. 在27℃下在含有5%胎牛血清(或FBS,PAA)的Insect Express Sf9/S2培养基(PAA)中培养Sf21细胞(本文称为"完全培养基")。
    2. 每4至6天,向新的25cm 2烧瓶中接种在5ml完全培养基中的1至1.25×10 6个活细胞。

  2. 产生在Sf21 细胞中表达MBP-MOS1融合蛋白的重组杆状病毒
    1. 将MBP-MOS1编码序列克隆到pVL-1392的SmaI限制性位点。
    2. 按照"Baculogold杆状病毒表达系统"制造商(BD Biosciences)的说明书( https://www.bdbiosciences.com/documents/Baculovirus_vector_system_manual.pdf )。
    3. 测定5至10批重组杆状病毒在感染的Sf21细胞中通过Western印迹产生重组MBP-MOS1的能力(https://www.sciencepub.net/researcher/0102/09_1259_yang_west_research0102.pdf )与抗MBP抗体(稀释1/10,000)。选择最有效的分离物用于进一步的蛋白质生产过程

  3. 在Sf21 细胞中产生MBP-MOS1融合蛋白
    1. 选择的杆状病毒原种用于感染Sf21细胞。
    2. 制备在30ml完全培养基中具有每个烧瓶6×10 7个活细胞的21个150cm 2烧瓶并在27℃温育直至细胞达到汇合见图1)。


      图1.融合的未感染的Sf21 细胞

    3. 然后用10ml不含FBS的培养基替换培养基,并用100μl高滴度重组杆状病毒储液(病毒滴度应为1至2×10 8 pfu/ml)感染细胞。
    4. 将细胞在27℃孵育3小时,然后加入5ml完全培养基(含5%FBS)。将细胞在27℃,黑暗中孵育3天
    5. 接种后2-3天检查感染迹象。 细胞应该放大(约2倍),并且应该可以看到大的细胞核(见图2)

      图2.感染的 Sf21 单元格

    6. 为了沉淀细胞,通过冲击轻轻地从单层中移出细胞,并将细胞悬浮液转移到适当大小的无菌离心管中。 以10,000×g离心细胞5分钟。 MBP-MOS1蛋白存在于细胞沉淀中,可以储存于-80℃或立即处理。

  4. MBP-MOS1融合蛋白提取粗提物
    1. 将沉淀在1×PBS中洗涤并合并,使得一个沉淀含有来自三个150cm 2烧瓶的细胞。
    2. 细胞质粗提取物(CCE)。
      1. 将每个沉淀重悬于3.5ml裂解缓冲液中。
      2. 将细胞在冰上裂解30分钟,然后在15,000×g和4℃下离心30分钟。 上清液(≈30ml)对应于细胞质粗提取物(CCE)。
      3. 通过Bradford测定(http://web.mnstate.edu/provost/bradfordproteinassayprotocol.pdf )。 保持CCE在冰上。
    3. 核原油提取物(NCE)
      1. 将每个沉淀重悬浮于0.75ml无NP40的裂解缓冲液中
      2. 在冰上超声处理(超声处理器,输出功率为100%,每次15秒的3个脉冲),并在10,000×g和4℃下离心10分钟。 上清液(≈6ml)对应于核粗提取物(NCE)。
      3. 蛋白质浓度通常为约5至10μg/μl(即总量约30至50mg)。 保持NCE在冰上。

  5. MBP-MOS1融合蛋白纯化
    先前获得的CCE和NCE可以单独处理或合并处理。 在下文中,将CCE和NCE汇集在一起,得到完整的细胞粗提取物。 MBP-MOS1可以通过两种方式纯化:使用1ml预装柱 或1ml批量的直链淀粉树脂。
    1. 预装柱
      1. 用至少5ml的结合缓冲液以0.5ml/min平衡柱子。
      2. 用NCE裂解缓冲液将粗提取物浓度调节至1mg/ml。 应用样品。
      3. 用至少10ml的结合缓冲液洗涤(直到流出物中没有物质出现)。 用5ml洗脱缓冲液洗脱,并收集成0.5ml级分。
      4. 在一个柱体积(约1ml)后,纯化的MBP-MOS1的峰在两至三个级分内洗脱,对应于在280nm具有最大UV吸光度的那些级分。
      5. 汇集阳性部分,并存储为50μl等分试样在-80℃
    2. 直链淀粉树脂
      1. 用至少5ml的结合缓冲液洗涤树脂。 向粗提取物中加入1ml树脂。
      2. 在4℃下温和混合孵育1小时。 以800×g离心并在4℃离心5分钟。 弃去上清液。
      3. 用至少10ml的结合缓冲液洗涤树脂沉淀。 以800×g离心并在4℃离心5分钟。 弃去上清液。 这将重复五次。
      4. 用1ml洗脱缓冲液洗脱MPB-MOS1; 以800×g离心并在4℃离心5分钟。 回收上清液(E1级分)。
      5. 洗脱可重复两次,得到E2和E3级分。
      6. 高度浓缩的级分(E1和通常E2)作为50μl等分试样储存在-80℃。
      7. 通过使用BSA作为标准的Bradford测定来测定阳性级分(来自两种方法)中的MBP-MOS1浓度。
      8. 从二十个F150cm 2感染的烧瓶中,根据感染的效率,预期约0.5至1μg/μl的1-2ml。

食谱

  1. 裂解缓冲液
    20mM Tris(pH7.6) 100 mM NaCl
    1 mM DTT
    1%NP40
    抗蛋白酶鸡尾酒(根据制造商的建议)
  2. 绑定缓冲区
    20mM Tris-盐酸(pH9) 100 mM NaCl
    1 mM DTT
  3. 洗脱缓冲液
    含有10mM麦芽糖的结合缓冲液

致谢

Dussassois-Montagne是法国政府(MRT)的博士研究生。 这项工作得到了Région中心(InhDDE项目),法国ANR(Elegineer项目),Tours大学和国家科学中心的支持。

参考文献

  1. Pflieger,A.,Jaillet,J.,Petit,A.,Auge-Gouillou,C.and Renault,S。(2014)。 Mos1转座过程中的目标捕获 J Biol Chem 289 (1):100-111。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jaillet, J., Dussaussois-Montagne, A., Renault, S. and Augé-Gouillou, C. (2014). Expression and Purification of the Eukaryotic MBP-MOS1 Transposase from Sf21 Insect Cells. Bio-protocol 4(20): e1262. DOI: 10.21769/BioProtoc.1262.
  2. Pflieger, A., Jaillet, J., Petit, A., Auge-Gouillou, C. and Renault, S. (2014). Target capture during Mos1 transposition. J Biol Chem 289(1): 100-111.
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