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Expression and Purification of the Arabidopsis E4 SUMO Ligases PIAL1 and PIAL2
拟南芥E4 SUMO连接酶PIAL1和PIAL2的表达与纯化   

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Abstract

The proteins PIAL1 (At1g08910) and PIAL2 (At5g41580) are members of the recently discovered group of plant E4 SUMO ligases. This protocol allows quick and simple expression of the recombinant proteins in Escherichia coli (E. coli) and subsequent affinity purification using a maltose binding protein (MBP) tag. The proteins can be used in SUMOylation reactions, where the MBP part of the protein can be detected with a commercially available antibody, or additional purification steps can be applied.

Keywords: Small ubiquitin-related modifier SUMO(小泛素相关修饰相扑), Protein modification(蛋白质的修饰), Plant proteins(植物蛋白), SUMO chain formation(泛素链的形成)

Materials and Reagents

  1. Rosetta DE3 pLysS strain (Merck KGaA, catalog number: 70956 )
  2. Vector pMAL-c2X (New England Biolabs, catalog number: N8076S ; the current version of the vector sold by NEB is pMAL-c5X, catalog number: N8108S )
  3. Peptone (ForMedium, catalog number: PEP03 )
  4. Yeast extract (ForMedium, catalog number: YEM03 )
  5. NaCl (Sigma-Aldrich, catalog number: S3014 )
  6. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  7. Ampicillin (Sigma-Aldrich, catalog number: A9518 )
  8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (GERBU Biotechnik GmbH, catalog number: 1010 )
  9. KCl (Sigma-Aldrich, catalog number: P5405 )
  10. Na2HPO4 (Sigma-Aldrich, catalog number: 71505 )
  11. KH2PO4 (Sigma-Aldrich, catalog number: P3786 )
  12. Aprotinin (Sigma-Aldrich, catalog number: A6279 ) (solution stored at 4 °C)
  13. Leupeptin (Sigma-Aldrich, catalog number: L2884 ) (stock solution of 1 mg/ml stored in aliquots at -20 °C)
  14. Amylose resin (New England Biolabs, catalog number: E8021S )
  15. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  16. Glycerol (GERBU Biotechnik GmbH, catalog number: 2006 )
  17. Ethylenediamine tetraacetic acid (EDTA) (GERBU Biotechnik GmbH, catalog number: 1034 )
  18. Tris (GERBU Biotechnik GmbH, catalog number: 1018 )
  19. Maltose (Merck KGaA, catalog number: 105911 )
  20. Ultrapure water (18.2 MΩ)
  21. Lysogeny broth (LB) (see Recipes)
  22. Phosphate buffered saline (PBS) (see Recipes)
  23. Column buffer (see Recipes)
  24. Elution buffer (see Recipes)

Equipment

  1. Orbital shaker at 37 °C
  2. Spectrophotometer
  3. Cooled centrifuge
  4. Bandelin Sonoplus sonicator with an MS 73 tapered probe (BANDELIN electronic GmbH & Co., model: GM70HD )
  5. PolyPrep Chromatography Columns (Bio-Rad Laboratories, AbD Serotec®, catalog number: 7311550 )

Procedure

  1. Expression
    1. The protein coding sequence for the E4 ligases was obtained from cDNA and inserted between the BamHI and SalI restriction sites in the vector pMAL-c2 as described in Tomanov et al. (2014). Thus, the sequence formed a continuous reading frame which was to be expressed as an N-terminal fusion to MBP (maltose binding protein).
    2. The plasmids were heat-shock transformed into the Rosetta DE3 pLysS strain.
    3.  A culture of 5 ml LB with 15 mg/L chloramphenicol and 100 mg/L ampicillin was inoculated and grown overnight at 37 °C with shaking at 200 rpm.
    4. On the next morning, 500 µl from the overnight culture were inoculated into 200 ml fresh LB with the same antibiotics.
    5. The culture was incubated at 37 °C with shaking at 200 rpm until OD600 reached 0.6 (around 3 h).
    6.  A 500 µl sample was withdrawn for electrophoretic analysis and stored at 4 °C. The culture was induced by adding IPTG to a final concentration of 1 mM.
    7. After three hours at 37 °C, another 500 µl sample was withdrawn and stored at 4 °C. The culture was harvested by centrifuging it at 4 °C and 4,500 x g for 20 min.
    8. The pellet was resuspended in PBS (pH 7.4) and centrifuged again at 4 °C and 4,500 x g for 20 min.
    9. The pellet was frozen at -20 °C overnight.

  2. Purification
    1. The frozen pellet was thawed on ice.
    2. Five milliliters of pre-cooled column buffer were added, together with aprotinin and leupeptin at a final concentration of 1 µg/ml.
    3. After 15 min on ice, the bacteria were lysed using a Bandelin Sonoplus HD70 sonicator with an MS 73 tapered probe (on ice). The ultrasound bursts were applied three times for 30 sec with intermediate cooling on ice and had 60% intensity, 50% on-time.
    4. The suspension was centrifuged for 20 min at 4,300 x g, 4 °C.
    5.  During the centrifugation, a BioRad PolyPrep Chromatography column (2 ml bed volume) was prepared by adding 200 µl amylose resin.
    6. The resin was washed with 3 column volumes (cv) water and equilibrated with 6 cv column buffer. The lower end of the column was capped and the centrifugation supernatant was added, along with 10 µg DNase I. Pre-incubation with DNase I is not necessary.
    7. The column was sealed and incubated for 30-60 min on a rotating wheel at 4 °C.
    8. The flow-through was collected and the column was washed with 24 cv column buffer.
    9. The protein was eluted with 3 x 1 cv elution buffer and glycerol was added to the eluates to a final concentration of 20% v/v.
    10. The purity and concentration of the eluted protein was checked with SDS-PAGE, and the fractions were snap-frozen in liquid nitrogen and kept at -80 °C.

Representative data


Figure 1. Representative results after expression and purification of MBP-PIAL2. Ten microliters of each fraction were mixed with 10 µl Laemmli sample buffer (Laemmli, 1970), boiled for 5 min at 95 °C and ran on a 10% SDS-PAGE gel. The non-induced (NI) and the induced (I) fraction were prepared by centrifuging 500 µl of the culture and adding 50 µl Laemmli sample buffer to the pellet. Twenty microliters were applied on the gel. Fraction names are as follows: NI - non-induced, I - induced, SN - supernatant (after sonication), FT - flow-through (after amylose resin incubation), W - wash, E - elution. Free MBP is visible in the induced fraction already and is likely a consequence of an in vivo proteolytic event.

Notes

  1. A more detailed explanation of SUMOylation and the enzymes involved is described by Tomanov et al. (2014).
  2. Judging from the usual quality of NEB products, there should not be any problems arising from using the current version of the pMAL vector.
  3. PBS can be prepared as a 10x stock; needs to be filtered before use.
  4. The column buffer can be stored for up to one month at 4 °C. The elution buffer can be stored up to six months at -20 °C. Freshly prepared buffers allow for the best results.
  5. Both protein fusions (MBP-PIAL1 and MBP-PIAL2) are soluble and retain their activity for in vitro SUMOylation for several months when stored at -80 °C. The pMAL-c2X vector contains a factor Xa cleavage site, so the protein can be additionally purified for crystallography studies by an in-column digest.
  6. For Western blot detection, NEB offers an MBP-specific antibody with catalog number E8032S.

Recipes

  1. Lysogeny broth (LB) (autoclaved)
    1 % w/v peptone
    0.5 % w/v NaCl
    0.5 % w/v yeast extract
    pH 7.2
  2. Phosphate buffered saline (PBS) (sterile filtered)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    pH 7.4
  3. Column buffer (sterile filtered)
    20 mM Tris
    200 mM NaCl
    1 mM EDTA
    pH 7.4
  4. Elution buffer (sterile filtered)
    20 mM Tris
    200 mM NaCl
    1 mM EDTA
    10 mM maltose
    pH 7.4

Acknowledgment

Work in A.B.´s laboratory was supported by the Austrian Science Fund FWF (grant P25488-B22).

References

  1. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  2. Tomanov, K., Zeschmann, A., Hermkes, R., Eifler, K., Ziba, I., Grieco, M., Novatchkova, M., Hofmann, K., Hesse, H. and Bachmair, A. (2014). Arabidopsis PIAL1 and 2 promote SUMO chain formation as E4-type SUMO ligases and are involved in stress responses and sulfur metabolism. Plant Cell 26(11): 4547-4560.

简介

蛋白质PIAL1(At1g08910)和PIAL2(At5g41580)是最近发现的植物E4 SUMO连接酶的成员。 该方案允许在大肠杆菌(大肠杆菌)中快速和简单地表达重组蛋白,并随后使用麦芽糖结合蛋白(MBP)标签进行亲和纯化。 蛋白质可用于SUMO化反应中,其中蛋白质的MBP部分可用市售的抗体检测,或可应用额外的纯化步骤。

关键字:小泛素相关修饰相扑, 蛋白质的修饰, 植物蛋白, 泛素链的形成

材料和试剂

  1. Rosetta DE3 pLysS菌株(Merck KGaA,目录号:70956)
  2. 载体pMAL-c2X(New England Biolabs,目录号:N8076S;由NEB销售的载体的当前版本是pMAL-c5X,目录号:N8108S)
  3. 蛋白胨(ForMedium,目录号:PEP03)
  4. 酵母提取物(ForMedium,目录号:YEM03)
  5. NaCl(Sigma-Aldrich,目录号:S3014)
  6. 氯霉素(Sigma-Aldrich,目录号:C0378)
  7. 氨苄青霉素(Sigma-Aldrich,目录号:A9518)
  8. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(GERBU Biotechnik GmbH,目录号:1010)
  9. KCl(Sigma-Aldrich,目录号:P5405)
  10. Na 2 HPO 4(Sigma-Aldrich,目录号:71505)
  11. KH sub 2 PO 4(Sigma-Aldrich,目录号:P3786)
  12. 抑肽酶(Sigma-Aldrich,目录号:A6279)(溶液贮存在4℃)
  13. 亮肽素(Sigma-Aldrich,目录号:L2884)(1mg/ml的储备液以等分试样在-20℃下储存)
  14. 直链淀粉树脂(New England Biolabs,目录号:E8021S)
  15. DNase I(Roche Diagnostics,目录号:10104159001)
  16. 甘油(GERBU Biotechnik GmbH,目录号:2006)
  17. 乙二胺四乙酸(EDTA)(GERBU Biotechnik GmbH,目录号:1034)
  18. Tris(GERBU Biotechnik GmbH,目录号:1018)
  19. 麦芽糖(Merck KGaA,目录号:105911)
  20. 超纯水(18.2MΩ)
  21. 溶菌酶肉汤(LB)(参见食谱)
  22. 磷酸盐缓冲盐水(PBS)(见Recipes)
  23. 列缓冲区(参见配方)
  24. 洗脱缓冲液(见配方)

设备

  1. 37°C的轨道摇床
  2. 分光光度计
  3. 冷却离心机
  4. 带有MS 73锥形探针(BANDELIN electronic GmbH& Co.,型号:GM70HD)的Bandelin Sonoplus超声波仪
  5. PolyPrep色谱柱(Bio-Rad Laboratories,AbD Serotec ,目录号:7311550)

程序

  1. 表达式
    1. 从cDNA获得E4连接酶的蛋白质编码序列   并插入载体中的BamHI和SalI限制性位点之间   pMAL-c2,如Tomanov等人(2014)中所述。 因此,序列 形成连续读框,其将被表示为 与MBP(麦芽糖结合蛋白)的N-末端融合
    2. 将质粒热休克转化到Rosetta DE3 pLysS菌株中
    3.   5毫升LB与15毫克/升氯霉素和100毫克/升的文化 接种氨苄青霉素并在37℃下振荡生长过夜 200 rpm。
    4. 第二天早上,将来自过夜培养物的500μl接种到具有相同抗生素的200ml新鲜LB中
    5. 将培养物在37℃下以200rpm振摇温育,直到OD 600达到0.6(约3小时)。
    6.  取出500μl样品用于电泳分析并储存 在4℃。 通过加入IPTG至终浓度诱导培养物   为1mM。
    7. 在37℃下3小时后,再加入500μl样品 取出并在4℃下储存。 通过离心收获培养物 它在4℃和4500xg下20分钟
    8. 将沉淀重悬浮于PBS(pH 7.4)中,并在4℃和4,500xg下再次离心20分钟。
    9. 将沉淀在-20℃下冷冻过夜。

  2. 净化
    1. 将冷冻的沉淀在冰上解冻。
    2. 五毫升 预冷柱缓冲液,以及抑肽酶和 亮肽素,终浓度为1μg/ml
    3. 15分钟后 冰,使用Bandelin Sonoplus HD70超声波仪裂解细菌 带有MS 73锥形探头(在冰上)。 施加超声波脉冲   三次,30秒,在冰上中间冷却,并且具有60% 强度,50%准时。
    4. 将悬浮液在4,300×g,4℃下离心20分钟。
    5.  在离心过程中,使用BioRad PolyPrep色谱柱(2 ml床体积)通过加入200μl直链淀粉树脂来制备。
    6. 的 树脂用3柱体积(cv)水洗涤并用6平衡   cv列缓冲区。 将柱的下端加盖 离心上清液与10μgDNase I一起加入。 不需要与DNA酶I预孵育。
    7. 将柱密封并在4℃下在旋转轮上温育30-60分钟
    8. 收集流出物并用24cv柱缓冲液洗涤柱。
    9. 用3×1 cv洗脱缓冲液洗脱蛋白质,并用甘油洗脱 加到洗脱液中至终浓度为20%v/v
    10. 的 检查洗脱蛋白的纯度和浓度 SDS-PAGE,并将级分在液氮中快速冷冻并保存 -80℃。

代表数据


图1.表达和纯化MBP-PIAL2后的代表性结果将10μl每种级分与10μlLaemmli样品缓冲液(Laemmli,1970)混合,在95℃煮沸5分钟,然后在10%SDS-PAGE凝胶上。通过离心500μl培养物并向沉淀物中加入50μlLaemmli样品缓冲液来制备非诱导(NI)和诱导(I)级分。将20微升施加在凝胶上。组分名称如下:NI-非诱导的,I-诱导的,SN-上清液(超声处理后),FT-流通(在直链淀粉树脂培养后),W-洗涤,E-洗脱。游离MBP在诱导级分中已经可见,并且可能是体内蛋白水解事件的结果。

笔记

  1. 关于SUMO化和所涉及的酶的更详细的解释由Tomanov等人(2014)描述。
  2. 从NEB产品的通常质量来看,使用当前版本的pMAL载体不会出现任何问题。
  3. PBS可以制备为10x原液;需要在使用前过滤。
  4. 柱缓冲液可以在4℃下储存长达一个月。洗脱缓冲液可在-20°C下储存6个月。新鲜制备的缓冲液有最好的效果。
  5. 当储存在-80℃时,两种蛋白质融合物(MBP-PIAL1和MBP-PIAL2)都是可溶的并且保留其体外的SUMO化几个月的活性。 pMAL-c2X载体含有因子Xa切割位点,因此可以通过柱内消化来进一步纯化蛋白质用于晶体学研究。
  6. 对于Western印迹检测,NEB提供了目录号为E8032S的MBP特异性抗体

食谱

  1. 溶菌酶肉汤(LB)(高压灭菌)
    1%w/v蛋白胨 0.5%w/v NaCl
    0.5%w/v酵母提取物
    pH 7.2
  2. 磷酸盐缓冲盐水(PBS)(无菌过滤)
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    pH 7.4
  3. 柱缓冲液(无菌过滤)
    20 mM Tris
    200 mM NaCl
    1mM EDTA
    pH 7.4
  4. 洗脱缓冲液(无菌过滤)
    20 mM Tris
    200 mM NaCl
    1mM EDTA
    10mM麦芽糖 pH 7.4

确认

在奥地利实验室的工作由奥地利科学基金会FWF(授予P25488-B22)支持。

参考文献

  1. Laemmli,U.K。(1970)。 在噬菌体T4头部装配过程中切割结构蛋白。 自然 227(5259):680-685
  2. Tomanov,K.,Zeschmann,A.,Hermkes,R.,Eifler,K.,Ziba,I.,Grieco,M.,Novatchkova,M.,Hofmann,K.,Hesse,H.and Bachmair, 2014)。 拟南芥PIAL1和2促进SUMO链形成为E4型SUMO连接酶,并参与胁迫反应和 硫代谢。植物细胞 26(11):4547-4560
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引用:Tomanov, K. and Bachmair, A. (2015). Expression and Purification of the Arabidopsis E4 SUMO Ligases PIAL1 and PIAL2. Bio-protocol 5(15): e1544. DOI: 10.21769/BioProtoc.1544.
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