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Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins
融合His及StrepII标签的莱茵衣藻蛋白的原核表达及纯化   

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Abstract

This protocol describes the heterologous expression and purification of proteins related to anoxic hydrogen production of Chlamydomonas reinhardtii (Noth et al., 2013). For this, the bacterial expression hosts Escherichia coli BL21 (DE3) ΔiscR (Akhtar MK et al., 2008) and Clostridium acetobutylicum ATCC 824 are used, which are grown either aerobic or anaerobic with glucose. Two standard chromatographic methods for purification were applied using His- and StrepII-tagged proteins (Figure 1). All procedures have been performed in an anaerobic tent to avoid the access of oxygen.

Keywords: Fermentation(发酵), Protein Isolation(蛋白质分离), Hydrogenase(氢化酶), Iron Sulfur Cluster(铁硫簇), Chlamydomonas reinhardtii(莱茵衣藻)


Figure 1. Coomassie stained SDS-PAGE of purified, heterologously expressed proteins from C. reinhardtii. M: MW marker PageRuler Prestained Protein Ladder 10-170 kDa; a) purified PFR1 loaded onto a 10% SDS-polyacrylamidgel; b) purified [2Fe2S] ferredoxin (PetF) loaded onto a 15% SDS-polyacrylamidgel; c) purifiedhydrogenase (HydA1) loaded onto a 10% SDS-polyacrylamidgel. Different amounts of protein are loaded onto each gel.

Materials and Reagents

  1. Expression vector (pASK-IBA)
  2. LB medium (Lennox) (Carl Roth)
  3. Vogel Bonner minimal medium (homemade) (Vogel HJ et al., 1956)
  4. Thiamin hydrochlorid (Carl Roth)
  5. Resazurin (Riedel-de Haën)
  6. Immidazole (Alfa Aesar)
  7. Escherichia coli BL21 (DE3) ΔiscR
  8. Clostridium acetobutylicum ATCC 824
  9. Ni Sepharose 6 Fast Flow (GE Healthcare)
  10. Strep-Tactin Superflow (IBA Gmb)
  11. Ampicillin
  12. Anhydrotetracycline
  13. Glucose
  14. Sodium dithionite (laboratory reagent grade > 85%)
  15. Avidin (Affiland)
  16. Strep tactin
  17. Glycerol
  18. d-desthiobiotin (≥ 98%, TLC)
  19. Thiamine pyrophosphate
  20. PageRuler Prestained Protein Ladder 10-170 kDa (Thermo Fisher Scientific, catalog number: 26616 )
  21. 0.1 M Tris buffer (pH 8) (see Recipes)
  22. Pre-equilibrated gravity flow Ni-NTA (see Recipes)

Equipment

  1. Airtight vial
  2. Sonicator: Branson Sonifier 250 (Branson)
  3. Ultracentrifuge
  4. Anaerobic tent (1% H2, 99% N2) (Toepffer Lab Systems)
  5. 0.2 μm pore size sterile filter (Sarstedt AG & Co.)
  6. NanoDrop (Paqlab, Germany)
  7. Batch fermenter (Infors HT, CH)

Procedure

  1. Anaerobic expression of pyruvate: ferredoxin oxidoreductase (Noth et al., 2013)
    1. Electroporation (Sambrook et al., 2006) of 100 μl E. coli BL21 (DE3) ΔiscR (Akhtar et al., 2008) with ~100 ng expression vector (pASK-IBA).
    2. Inoculation of 200 ml LB and aerobic growth of a preculture over night at 37 °C (180 rpm).
    3. Inoculation of 4 L Vogel Bonner medium (8 x 500 ml; 2,000 ml Erlenmeyer flasks) supplemented with 100 μg/ml ampicillin, 50 μM thiamin hydrochlorid and 0.2 μM resazurin using 15 ml preculture each.
    4. Aerobic growth at 37 °C and 180 rpm until the culture reaches the anaerobic phase at A600 of 0.6. At that point, the redox indicator resazurin within the medium turns from blue to pink.
    5. Each 2 L of culture are induced by adding 0.2 μg/ml anhydrotetracycline and transferred into sterile 2 L Schott flasks containing 50 ml 20% glucose (5 g/L).
    6. Protein expression is carried out over night at 8 °C without stirring.
    7. Cells are anaerobically harvested by centrifugation for 20 min at 7,500 x g, resuspended in Tris-HCl (pH 8.0), 10% glycerol and stored at -20 °C until purification.

  2. Anaerobic purification of pyruvate: ferredoxin oxidoreductase (His-tag)
    1. For purification the pellet (2 L of culture) is thawed at room temperature and lysed by sonication while keeping the cells cooled on ice.
      Note: Five times for 30 sec; output, 25; Branson Sonifier 250.
    2. Sedimentation of cell debris at 200,000 x g for 60 min and 4 °C in an ultracentrifuge.
    3. The soluble fraction is filtered using a pore size of 0.2 μm to get rid of unwanted material which clogs the column.
    4. Then, the sample is loaded on a pre-equilibrated (100 mM Tris-HCl, pH 8.0, 10 mM imidazole, 0.5 mM thiamine pyrophosphate) gravity flow Ni-NTA fast-flow column with a bed volume of 4 ml.
    5. Protein purification is achieved via increasing the imidazole concentration from 10 to 20 mM during washing each with 40 ml buffer.
    6. The His-tagged PFR1 protein is eluted from the column with 10 ml buffer containing 100 mM imidazole. Nine elution fractions each 1.1 ml are collected.
    7. The protein concentration of the brownish main elution fractions 3 and 4 are immediately determined using A280.

  3. Aerobic expression of [2Fe2S] ferredoxins (Jacobs et al., 2009; Winkler et al., 2009) with minor changes
    1. E. coli BL21 (DE3) ΔiscR containing the expression plasmid pASK-IBA7-FDX is grown in Vogel Bonner minimal medium for 4 h after induction at A600 of 0.6.
    2. Cells are harvested, washed in Tris-HCl (pH 8.0), sedimented again and stored at -20 °C until purification.

  4. Anaerobic expression of HydA1 (Girbal et al., 2005; von Abendroth et al., 2008)
    1. Expression plasmid containing C. acetobutylicum ATCC 824 strain is grown in CGM-medium and a glucose concentration of 60 g/L anaerobically in a batch fermenter over night at 35-37 °C and 100 rpm.
    2. Cells are harvested in an anaerobic tent analog to E. coli, resuspended in Tris-HCl (pH 8.0), 10% glycerol containing 10 mM sodium dithionite and stored at -20 °C until purification.

  5. Anaerobic expression of bacterial 2[4Fe4S] ferredoxin analog to HydA1 (Girbal et al., 2005; von Abendroth et al., 2008, Noth et al., 2013)
    1. Expression plasmid containing C. acetobutylicum ATCC 824 strain is grown in CGM-medium and a glucose concentration of 60 g/L anaerobically in a batch fermenter over night at 35-37 °C and 100 rpm.
    2. Cells are harvested in an anaerobic tent analog to E. coli, resuspended in Tris-HCl (pH 8.0), 10% glycerol containing 10 mM sodium dithionite and stored at -20 °C until purification.
  6. Anaerobic purification of StrepII-tagged proteins (C-E)
    1. All buffers used contain 2 mM sodium dithionite.
    2. For purification the cell pellet is thawed at room temperature and lysed by sonication while keeping the cells cooled on ice.
      Note: Five times for 30 sec; output, 25; Branson Sonifier 250.
    3. Sedimentation of cell debris at 200,000 x g for 60 min and 4 °C in an ultracentrifuge.
    4. Supernatant (40 ml) is incubated for 1 hour with 3.5 mg Avidin (Stock 50 mg.ml-1) at 4 °C.
    5. The soluble fraction is filtered using a poresize of 0.2 μm to get rid of biotinylated, complexed proteins and unwanted material which clogs the column.
    6. Then, the filtered solution is loaded on a Tris-HCl (pH 8.0) equilibrated 2 ml strep tactin gravity flow column.
    7. The unbound proteins are washed from the column using 80 ml Tris-HCl (pH 8.0).
    8. Elution is performed with 10 ml Tris-HCl (pH 8.0), d-desthiobiotin (0.8 mg/ml) in fractions of 1 ml.

Recipes

  1. 0.1 M Tris buffer (pH 8) (1,000 ml)
    Mix 12.114 g of Tris base with 800 ml dH2O
    Add 100 ml Glycerol
    pH to 8 with HCl
    Add ddH2O to 1,000 ml
    Autoclave for 20 min at 121 °C
    Store at 4 °C
  2. Pre-equilibrated gravity flow Ni-NTA
    0.1 mM Tris-HCl (pH 8)
    10 mM imidazole
    0.5 mM thiamine pyrophosphate

Acknowledgments

Aerobic expression of [2Fe2S] ferredoxins was adapted from Jacobs et al. (2009). Anaerobic expression and purification of the 2[4Fe4S] bacterial type ferredoxin was done according to the previously published isolation of [FeFe]-Hydrogenase HydA1 from Chlamydomonas reinhardtii by Girbal et al. (2005) and von Abendroth et al. (2008), which is also presented here. Research on the pyruvate:ferredoxin oxidoreductase from C. reinhardtii was scientifically supported by Anja Hemschemeier and Thomas Happe.

References

  1. Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
  2. Akhtar, M. K. and Jones, P. R. (2008). Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 78(5): 853-862.
  3. Girbal, L., von Abendroth, G., Winkler, M., Benton, P. M., Meynial-Salles, I., Croux, C., Peters, J. W., Happe, T. and Soucaille, P. (2005). Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71(5): 2777-2781.
  4. Jacobs, J., Pudollek, S., Hemschemeier, A. and Happe, T. (2009). A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii. FEBS Lett 583(2): 325-329.
  5. Sambrook, J. and Russell, D. W. (2006). Transformation of E. coli by Electroporation. CSH Protoc 2006(1).
  6. Vogel, H. J. and Bonner, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218(1): 97-106.
  7. von Abendroth, G., Stripp, S., Silakov, A., Croux, C., Soucaille, P., Girbal, L. and Happe, T. (2008). Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum. Inter J Hydrogen Energy 33(21): 6076-6081.
  8. Winkler, M., Kuhlgert, S., Hippler, M. and Happe, T. (2009). Characterization of the key step for light-driven hydrogen evolution in green algae. J Biol Chem 284(52): 36620-36627.

简介

该方案描述了与赖氨酸衣藻的缺氧氢生产相关的蛋白质的异源表达和纯化(Noth等,2013)。 为此,使用大肠杆菌BL21(DE3)ΔiscR(Akhtar MK等人,2008)和丙酮丁醇梭菌ATCC 824的细菌表达,其用葡萄糖生长需氧或厌氧。 使用His和StrepII标记的蛋白质应用两种标准色谱纯化方法(图1)。 所有程序已在厌氧帐篷中进行,以避免氧气进入。

关键字:发酵, 蛋白质分离, 氢化酶, 铁硫簇, 莱茵衣藻


图1.来自C的纯化的异源表达的蛋白质的考马斯染色的SDS-PAGE。 M:MW marker PageRuler预染蛋白梯度10-170 kDa; a)装载到10%SDS-聚丙烯酰胺凝胶上的纯化的PFR1; b)装载到15%SDS-聚丙烯酰胺凝胶上的纯化的[2Fe 2 S]铁氧还蛋白(PetF) c)装载到10%SDS-聚丙烯酰胺凝胶上的纯化的氢化酶(HydA1)。 将不同量的蛋白质加载到每个凝胶上。

材料和试剂

  1. 表达载体(pASK-IBA)
  2. LB培养基(Lennox)(Carl Roth)
  3. Vogel Bonner基本培养基(自制)(Vogel HJ et al。,1956)
  4. 硫胺盐酸盐(Carl Roth)
  5. 刃天青(Riedel-deHaën)
  6. 咪唑(阿法埃莎)
  7. 大肠杆菌 BL21(DE3)ΔiscR
  8. <丙酮丙酸梭菌ATCC ATCC 824
  9. Ni Sepharose 6 Fast Flow(GE Healthcare)
  10. Strep-Tactin Superflow(IBA Gmb)
  11. 氨苄西林
  12. 脱水四环素
  13. 葡萄糖
  14. 连二亚硫酸钠(实验室试剂级> 85%)
  15. 抗生素(Affiland)
  16. 链球菌素
  17. 甘油
  18. d-desthiobiotin(≥98%,TLC)
  19. 焦磷酸硫胺素
  20. PageRuler预染蛋白梯度10-170 kDa(Thermo Fisher Scientific,目录号:26616)
  21. 0.1 M Tris缓冲液(pH 8)(参见配方)
  22. 预平衡重力流Ni-NTA(参见配方)

设备

  1. 气密管
  2. 超声波仪:Branson Sonifier 250(Branson)
  3. 超速离心机
  4. 厌氧性帐篷(1%H 2 O,99%N 2)(Toepffer Lab Systems)
  5. 0.2μm孔径无菌过滤器(Sarstedt AG& Co.)
  6. NanoDrop(Paqlab,德国)
  7. 分批发酵罐(Infors HT,CH)

程序

  1. 丙酮酸的厌氧表达:铁氧还蛋白氧化还原酶(Noth et al。,2013)
    1. 电穿孔(Sambrook等人,2006)100μlE。 (pASK-IBA)的大肠杆菌 BL21(DE3)ΔiscR(Akhtar等人,
    2. 在37℃(180rpm)下接种200ml LB和预培养物有氧生长过夜
    3. 用每次15ml预培养物接种补充有100μg/ml氨苄青霉素,50μM盐酸盐和0.2μM刃天青的4L Vogel Bonner培养基(8×500ml; 2,000ml锥形瓶)。
    4. 在37℃和180rpm下需氧生长,直到培养物在A 600的0.6达到厌氧阶段。 此时,介质中的氧化还原指示剂刃天青从蓝色变为粉红色
    5. 通过加入0.2μg/ml脱水四环素诱导每2L培养物,并转移到含有50ml 20%葡萄糖(5g/L)的无菌2L Schott烧瓶中。
    6. 蛋白质表达在8℃下搅拌过夜
    7. 通过在7,500×g离心20分钟来厌氧收获细胞,重悬于Tris-HCl(pH 8.0),10%甘油中并储存在-20℃直至纯化。
  2. 丙酮酸的厌氧纯化:铁氧还蛋白氧化还原酶(His-标签)
    1. 为了纯化,将沉淀(2L培养物)在室温下解冻,并通过超声处理裂解,同时保持细胞在冰上冷却。
      注意:5次30秒; 输出,25; Branson Sonifier 250。
    2. 在超速离心机中将细胞碎片在200,000×g下沉淀60分钟和4℃。
    3. 使用0.2μm的孔径过滤可溶性级分以除去堵塞柱的不想要的物质
    4. 然后,将样品加载到预平衡的(100mM Tris-HCl,pH 8.0,10mM咪唑,0.5mM焦磷酸硫胺素)重力流动Ni-NTA快速流动柱上,柱床体积为4ml。 >
    5. 蛋白质纯化通过在用40ml缓冲液洗涤期间将咪唑浓度从10mM增加至20mM来实现
    6. 用含有100mM咪唑的10ml缓冲液从柱上洗脱His-标记的PFR1蛋白。 收集9个洗脱级分,每次1.1ml
    7. 使用A 280立即测定棕色主洗脱级分3和4的蛋白质浓度。

  3. [2Fe2S]铁氧还蛋白的好氧表达(Jacobs等人,2009; Winkler等人,2009)
    1. E。 含有表达质粒pASK-IBA7-FDX的大肠杆菌BL21(DE3)ΔiscR在Vermel Bonner基本培养基中生长4小时,在A 600 600 。
    2. 收获细胞,在Tris-HCl(pH8.0)中洗涤,再次沉降并储存在-20℃直至纯化
  4. HydA1的厌氧表达(Girbal等人,2005; von Abendroth等人,2008)
    1. 含有C的表达质粒。 丙酮丁醇ATCC 824菌株在分批发酵罐中于35-37℃和100rpm下在CGM培养基和葡萄糖浓度为60g/L的条件下生长过夜。
    2. 在厌氧条件下将细胞收获在大肠杆菌中,重悬于Tris-HCl(pH 8.0),含有10mM连二亚硫酸钠的10%甘油中,并储存在-20℃直至纯化。

  5. 细菌2 [4Fe4S]铁氧还蛋白类似物对HydA1的厌氧表达(Girbal等,2005; von Abendroth等,2008,Noth等,/em>,2013)
    1. 含有C的表达质粒。 丙酮丁醇梭菌ATCC 824菌株在分批发酵罐中在35-37℃和100rpm下在CGM培养基和葡萄糖浓度为60g/L的厌氧条件下生长过夜。 >
    2. 在类似于E的厌氧帐篷中收获细胞。 大肠杆菌,再悬浮于Tris-HCl(pH 8.0),含有10mM连二亚硫酸钠的10%甘油中,并储存在-20℃直至纯化。
  6. 厌氧纯化StrepII标记蛋白(C-E)
    1. 所有使用的缓冲液含有2mM连二亚硫酸钠
    2. 为了纯化,将细胞沉淀在室温下解冻并通过超声处理裂解,同时保持细胞在冰上冷却。
      注意:5次30秒; 输出,25; Branson Sonifier 250。
    3. 在超速离心机中将细胞碎片在200,000×g下沉淀60分钟和4℃。
    4. 在4℃下将上清液(40ml)与3.5mg抗生物素蛋白(储备液50mg缓冲液 ml -1)孵育1小时。
    5. 使用0.2μm的孔径过滤可溶性级分以除去生物素化的复合蛋白质和阻塞柱的不想要的物质。
    6. 然后,将过滤的溶液装载在经平衡的2ml链霉亲和流动柱的Tris-HCl(pH 8.0)上。
    7. 用80ml Tris-HCl(pH8.0)从柱上洗去未结合的蛋白质
    8. 用10ml Tris-HCl(pH 8.0),d-脱硫生物素(0.8mg/ml升)以1ml的级分进行洗脱。

食谱

  1. 0.1M Tris缓冲液(pH8)(1,000ml) 将12.114g Tris碱与800ml dH 2 O混合 加入100毫升甘油
    用盐酸将pH调至8 将ddH <2> O添加到1,000 ml
    在121°C高压灭菌20分钟
    存储在4°C
  2. 预平衡重力流Ni-NTA
    0.1mM Tris-HCl(pH8)
    10mM咪唑 0.5mM焦磷酸硫胺素

致谢

[2Fe2S]铁氧还蛋白的好氧表达来自Jacobs等人(2009)。根据Girbal等人先前公开的来自莱茵衣藻的[FeFe] - 氢化酶HydA1的分离进行2 [4Fe4S]细菌类型铁氧还蛋白的厌氧表达和纯化。 (2005)和von Abendroth等人(2008),其也在这里给出。丙酮酸:来自C的铁氧还蛋白氧化还原酶的研究。 reinhardtii 由Anja Hemschemeier和Thomas Happe科学支持。

参考文献

  1. Noth,J.,Krawietz,D.,Hemschemeier,A.and Happe,T。(2013)。 丙酮酸:铁氧还蛋白氧化还原酶偶联于莱茵衣藻中与光无关的氢产生。 J Biol Chem 288(6):4368-4377。
  2. Akhtar,M.K.and Jones,P.R。(2008)。 缺失iscR可刺激大肠杆菌中的重组梭菌Fe-Fe氢化酶活性和H2累积 BL21(DE3)。 Appl Microbiol Biotechnol 78(5):853-862。
  3. Girbal,L.,von Abendroth,G.,Winkler,M.,Benton,P.M.,Meynial-Salles,I.,Croux,C.,Peters,J.W.,Happe,T.and Soucaille, 丙酮丁醇梭菌中的同源和异源过表达和纯化的梭菌和藻类的表征只有Fe的氢化酶具有高比活性。 Appl Environ Microbiol 71(5):2777-2781。
  4. Jacobs,J.,Pudollek,S.,Hemschemeier,A.and Happe,T。(2009)。 莱茵衣藻中的一种小说,厌氧诱导的铁氧还蛋白。 FEBS Lett 583(2):325-329。
  5. Sambrook,J。和Russell,D.W。(2006)。 的转换。大肠杆菌。 CSH Protoc 2006(1)。
  6. Vogel,H.J.and Bonner,D.M。(1956)。 大肠杆菌的乙酰吗啉酶:部分纯化和一些特性。 J Biol Chem 218(1):97-106
  7. von Abendroth,G.,Stripp,S.,Silakov,A.,Croux,C.,Soucaille,P.,Girbal,L.and Happe,T。(2008)。 在丙酮丁醇梭菌中具有高比活性的[FeFe]氢化酶的优化过表达 em>。 Inter J Hydrogen Energy 33(21):6076-6081。
  8. Winkler,M.,Kuhlgert,S.,Hippler,M.and Happe,T。(2009)。 绿色藻类中光驱氢析出的关键步骤的特征。 J Biol Chem 284(52):36620-36627。
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Copyright: © 2013 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. Noth, J. (2013). Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins . Bio-protocol 3(17): e881. DOI: 10.21769/BioProtoc.881.
  2. Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
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