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Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP
红球菌属1CP中的氯代粘康酸内酯脱卤素酶CLcF的纯化和结晶化   

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Abstract

The protocol describes the generation of variants of chloromuconolactone dehalogenase from Rhodococcus opacus (R. opacus) 1CP. ClcF is a multimeric protein, which catalyses the dechlorination of 5-chloromuconolactone to cis-dienelactone in the 3-chlorocatecholic acid degradation pathway. The protocol describes the workflow for the purification and subsequent crystallization of the enzyme. The used workflow and the described techniques could be easily adapted to any other protein/enzyme intended to be crystallized by the potential user for subsequent structure determination. The protocol does not involve expensive specialized equipment which allows the use in standard laboratories not specially dedicated to macromolecular crystallography.

Keywords: Crystallisation(结晶), Protein purification(蛋白纯化), Column chromatography(柱色谱法), Recombinant protein expression(重组蛋白的表达), Biochemistry(生物化学)

Materials and Reagents

  1. Escherichia coli (E. coli) BL21 (DE3)-CP-RIL (Stratagene, catalog number: 230245 )
  2. E.coli DH5α (Life Technologies, catalog number: 18258012 )
  3. wtClcF plasmid (not commerically available)
  4. Forward Primer (not commerically available)
  5. Reverse Primer (not commerically available)
  6. dNTP-Mix (20 mM) (Thermo Fisher Scientific, catalog number: AB-0196
  7. Pfu-Ultra (2.5 U/µl) (Agilent, catalog number: 600385 )
  8. DpnI (Thermo Fisher Scientific, catalog number: ER1701 )
  9. LB medium
  10. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth, catalog number: CN08.1 )
  11. Tris base (Carl Roth, catalog number: 5429.1 )
  12. DNase (Sigma-Aldrich, catalog number: DN25-100MG )
  13. Ethylene glycol
  14. MgCl2 (Carl Roth, catalog number: 2189.2 )
  15. PEG 3350 (Sigma-Aldrich, catalog number: 202440-250G )
  16. Bis-Tris (Carl Roth, catalog number: 9140.1 )
  17. Liquid nitrogen
  18. Lysis buffer/Ion exchange (IEX) buffer A/Hydrophobic interaction chromatography (HIC) buffer A (see Recipes)
  19. HIC High salt buffer (see Recipes)
  20. Wash buffer (see Recipes)
  21. Crystallization buffers (see Recipes)

Equipment

  1. Standard laboratory equipment
  2. Nanodrop device (Thermo Fisher Scientific)
  3. CrystalQuickTM 96 well crystallization plate Greiner 609101(Jena Bioscience, catalog number: CPL-118S )
  4. Linbro crystallization plate (Jena Bioscience, catalog number: CPL-101S )
  5. 22 mm circular cover slides (siliconized) (Jena Bioscience, catalog number: CSL-106 )
  6. Bayer silicone grease (Jena Bioscience, catalog number: CGR-101 )
  7. Äkta purification system (GE Healthcare)
  8. HiTrap Q sepharose column (1 ml) (GE Healthcare, catalog number: 17-5053-01 )
  9. HiTrap phenyl-sepharose (1 ml) (GE Healthcare, catalog number: 17-1351-01 )
  10. Stereo microscope (Leica Microsystems, model: MZ12 )
  11. Eppendorf tubes
  12. Pipetting robot (if available)
  13. 37 °C incubator
  14. Centrifuge
  15. Vivaspin concentrator (MWCO 30 kDa) (EMD Millipore)

Procedure

  1. Mutation and overexpression
    Site directed mutagenesis by whole plasmid PCR was essentially carried out as described by Weiner et al. (1994).
    1. The mutagenesis-PCR was setup as followed:
      1.25 µl wtClcF plasmid (6 kbp) (20 ng/µl)
      1.25 µl forward Primer (100 ng/µl or 10 µM) with the respective mutation site
      1.25 µl reverse Primer (100 ng/µl or 10 µM) with the respective mutation site
      5 µl dNTP-Mix (2.5 mM)
      5 µl Pfu-Ultra buffer
      1 µl Pfu-Ultra 2.5 U/µl
      35.25 µl Water
      PCR-Program:

      Temperature
      Time

      95 °C
      30 sec

      95 °C
      30 sec
      18 cycles
      55 °C
      1 min
      72 °C
      1 min/kbp plasmid


    2. At the end the reaction was supplemented with 1/10 volume of 10x DpnI buffer and 10 U DpnI were added. The reaction was incubated at 37 °C for 1 h.
    3. Afterwards 1.25 µl of this mixture was transformed in ultracompetent E. coli DH5α.
    4. Sequence integrity has to be proved by sequencing.
    5. For expression purposes the plasmid with the codon sequence of ClcF or its mutants was transformed in E. coli BL21 (DE3)-CP-RIL.
    6.  For large scale protein production an overnight culture was setup with a colony from the transformation plate. The culture was incubated at 37 °C and 250 rpm for 16 h in LB medium.
    7. Afterwards 1 L LB medium was inoculated with the overnight culture in a ratio of 1/100 and further incubated at 37 °C and 250 rpm.
    8. If the culture reached an optical density (OD600 nm) of 0.6 (app. 2-2.5 h) the protein production was induced by adding 1 M IPTG stock solution to a final concentration of 1 mM. The culture was further incubated for 4 h at 30 °C.
    9. After 4 h induction the cells were harvested at 4,500 x g for 25 min at 4 °C.
    10. The resulting pellet was resuspended in app. 20 ml wash buffer and again pelleted at 4,500 x g for 45 min at 4 °C.
    11. The resulting pellet was stored at -20 °C until further usage.

  2. Purification
    To get protein of sufficient puritiy ≤ 95 % (estimated from a SDS-gel) a three step strategy including a heat precipitation followed by a two step column chromatography was necessary.
    1. The cell pellet from 1 liter culture volume was resuspended in 25 ml lysis buffer. A 2 M MgCl2 stock solution was added to a final concentration of 3 mM and 0.5 U DNase were used to the digest DNA.
    2. Cells were disrupted using a precooled French Press at 1,500 Psi. The cells were passed three times through the French Press to ensure complete lysis.
    3. The cell debris was removed by centrifugation at 50,000 x g for 1 h.
    4. The supernatant was heated up to 65 °C for 10 min in a water bath and afterwards cooled down on ice.
    5. The denatured protein was removed by centrifugation at 50,000 x g for 1 h.
    6. The resulting supernatant (app. 25 ml) was applied on a Q-sepharose, preequilibrated with IEX-A buffer.
    7. The protein was applied on the column and the ClcF containing flowthrough (app. 30 ml) was collected.
    8. Solid ammonium sulfate (AMS) was added to a final concentration of 1.6 M to the flowthrough of the previous step (app. 6 g).
    9. The resulting solution was applied on a Phenyl-sepharose preequilibrated with HIC buffer. The protein was eluted with a linear gradient to 0 M AMS over 20 column volumes by means of an Aekta chromatography system. ClcF containing fractions were identified by activity assay as described in the underlying paper (Roth et al., 2013).
    10. ClcF containing fractions were pooled and dialyzed extensively against 250 times the protein volume of 25 mM Tris (pH 7.5) at 4 °C. The dialysis buffer is changed once after 12 h.
    11. Prior crystallization, the protein was concentrated to 10 mg/ml (determined using the Bradford assay) with a VivaSpin concentrator with a MWCO of 30,000 kDa at 4,000 x g. Aliquots of the protein were directly frozen at -80 °C till further usage.

  3. Crystallization
    1. Based on the recipes of the commercial or published screens (e.g. sparse matrix, grid screen), all necessary buffer solution were prepared as stock solutions at a concentration of 1 M at the relevant pH [Jancarik and Kim, 1991; Brzozowski and Walton, 2001; Hampton Research (see Reference 5); Jena Bioscience (see Reference 6)].
    2. All salt solutions and precipitant solutions were prepared as stock solutions at the highest concentration possible and filtered if applicable. For ClcF, 1 M Bis-Tris solutions with a pH 6.0 and 7.0 with a spacing of 0.2 pH units are produced (pH adjusted with NaOH/HCl). Furthermore a 2 M MgCl2-solution and a solution of 50 % (w/v) PEG 3350 are necessary. All solutions are prepared with double distilled water.
    3. The conditions of the respective screens were generated by mixing the appropriate stock solutions, previously prepared, in an Eppendorf tube.
    4. A screen was setup by a pipetting robot with a reservoir of 90 µl in a Greiner Crystalquick 96 well crystallization plate.
    5. Drops were setup by pipetting 0.2 µl reservoir solution and 0.2 µl protein solution respectively together. No further mixing is required. The plates are stored afterwards at 19 °C. The plates were then regularly examined (in the first week every day, afterwards weekly) for crystal formation. Crystals usually appears between one day and up to 3 months, but occasionally even longer periods might be necessary to obtain crystals. If a promising condition (crystals, microcrystals or crystalline precipitate) was identified a fine screen was setup in a hanging drop 24 well format usually by varying the pH around the initial condition in a range of ± 1.5 pH units and the precipitant concentration within an applicable range (Figure 1).


      Figure 1. Typical scheme for a 2-dimensional 24 well grid screen, varying pH and precipitant concentration at the same time

    6. The reservoir was prepared as described and drops were setup with a total drop volume of 2 µl by mixing 1 µl protein with 1 µl reservoir over a reservoir of 500 µl in a 24 well Linbro crystallization plate.
    7. For orthorhombic crystals of ClcF the screen, with a constant concentration of 0.4 M MgCl2 in all conditions, is shown in Figure 2.


      Figure 2. Typical screen for orthorhombic ClcF crystals, varying pH and precipitant concentration at the same time. In all conditions is MgCl2 in a final concentration of 0.4 M.

    8. For a complex the protein was premixed with 10 mM substrate (5-chloromuconolactone), dissolved in a concentration of 1 M in water at pH 7 (adjusted with NaHCO3). No elongated incubation period is required prior crystallization. The setup of the crystallization plate was carried out as described.


      Figure 3. Promising crystalline material within protein precipitate as starting point for further fine screens (left) and typical single crystals of ClcF (right). Further pictures could be found on the Hampton Research website (see Reference 5).

  4. Cryoprotection
    1. A condition with good looking crystals was chosen and the reservoir solution was prepared with 2% elevated precipitant concentration supplemented with 25% ethylene glycol.
    2. Subsequently small amounts, 0.1 to 0.2 µl of the cryosolution was added to the drop and the drop solution slowly exchanged against the cryosolution.
    3. In the case of the complex the cryosolution contained the precipitant in a final concentration of 25% plus the ligand in a concentration of 10 mM. No additional ethylene glycol is required.
    4. Finally the crystals were fished with a nylon loop and plunged into liquid nitrogen.

Recipes

  1. Lysis buffer/Ion exchange (IEX) buffer A/Hydrophobic interaction chromatography (HIC) buffer A
    25 mM Tris-HCl (pH 7.5)
  2. HIC High salt buffer
    25 mM Tris-HCl (pH 7.5) (final pH after all ingredients are added)
    1.6 M ammonium sulphate
  3. Wash buffer
    50 mM Tris (pH 7.0)
    150 mM NaCl
  4. Crystallization buffers
    pH adjusted with HCl or NaOH

Acknowledgments

The authors used the following standard protocols developed by the mentioned researchers. The Bradford Assay as described in Bradford (1976). The SDS-gel electrophoresis as described in Laemmli (1970). Standard recipes for media and standard buffers are adapted from: “Molecular Cloning.” Green, M. R. and Sambrook, J. Cold Spring Harbor Laboratory Press.

References

  1. Brzozowski, A. M. and Walton, J. (2001). Clear strategy screens for macromolecular crystallization. J Appl Crystallogr 34(2): 97-101.
  2. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  3. Hampton Research. www.hamptonresearch.com 
  4. Jancarik, J. and Kim, S. H. (1991). Sparse matrix sampling: a screening method for crystallization of proteins. J Appl Crystallogr 24(4): 409-411. 
  5. Jena Bioscience. www.jenabioscience.com
  6. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.   
  7. Roth, C., Gröning, J. A., Kaschabek, S. R., Schlömann, M. and Sträter, N. (2013). Crystal structure and catalytic mechanism of chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP. Mol Microbiol 88(2): 254-267.
  8. Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E. and Bauer, J. C. (1994). Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151(1-2): 119-123.

简介

该方案描述了来自不透明红球菌(emocus)( R。opacus )1CP的氯木聚糖内酯脱卤酶变体的产生。 ClcF是多聚体蛋白,其催化5-氯代粘康酸内酯在3-氯代儿茶酸降解途径中脱氯成顺式 - 二内酯。 该方案描述了酶的纯化和随后的结晶的工作流程。 所使用的工作流程和所描述的技术可以容易地适用于潜在用户为了随后的结构确定而被结晶的任何其它蛋白质/酶。 该协议不涉及昂贵的专用设备,其允许在不专门用于大分子晶体学的标准实验室中使用。

关键字:结晶, 蛋白纯化, 柱色谱法, 重组蛋白的表达, 生物化学

材料和试剂

  1. 大肠杆菌(大肠杆菌)BL21(DE3)-CP-RIL(Stratagene,目录号:230245)
  2. E.coli DH5α(Life Technologies,目录号:18258012)
  3. wtClcF质粒(不可商业获得)
  4. 正向引物(不是商业上可用的)
  5. 反向引物(不是商业上可用的)
  6. dNTP-Mix(20mM)(Thermo Fisher Scientific,目录号:AB-0196)
  7. Pfu-Ultra(2.5U /μl)(Agilent,目录号:600385)
  8. DpnI(Thermo Fisher Scientific,目录号:ER1701)
  9. LB培养基
  10. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Carl Roth,目录号:CN08.1)
  11. Tris碱(Carl Roth,目录号:5429.1)
  12. DNase(Sigma-Aldrich,目录号:DN25-100MG)
  13. 乙二醇
  14. MgCl 2(Carl Roth,目录号:2189.2)
  15. PEG 3350(Sigma-Aldrich,目录号:202440-250G)
  16. Bis-Tris(Carl Roth,目录号:9140.1)
  17. 液氮
  18. 裂解缓冲液/离子交换(IEX)缓冲液A /疏水相互作用层析(HIC)缓冲液A(参见配方)
  19. HIC高盐缓冲液(见配方)
  20. 洗涤缓冲液(见配方)
  21. 结晶缓冲液(参见配方)

设备

  1. 标准实验室设备
  2. Nanodrop设备(Thermo Fisher Scientific)
  3. CrystalQuick TM 96孔结晶板Greiner 609101(Jena Bioscience,目录号:CPL-118S)
  4. Linbro结晶板(Jena Bioscience,目录号:CPL-101S)
  5. 22mm圆形盖玻片(硅化的)(Jena Bioscience,目录号:CSL-106)
  6. Bayer硅油(Jena Bioscience,目录号:CGR-101)
  7. Äkta纯化系统(GE Healthcare)
  8. HiTrap Q琼脂糖柱(1ml)(GE Healthcare,目录号:17-5053-01)
  9. HiTrap苯基 - 琼脂糖(1ml)(GE Healthcare,目录号:17-1351-01)
  10. 立体显微镜(Leica Microsystems,型号:MZ12)
  11. Eppendorf管
  12. 移液机器人(如果有)
  13. 37℃孵育器
  14. 离心机
  15. Vivaspin浓缩器(MWCO 30kDa)(EMD Millipore)

程序

  1. 突变和过表达
    通过整个质粒PCR的定点诱变基本上如Weiner等人(1994)所述进行。
    1. 诱变-PCR设置如下:
      1.25μlwt ClcF质粒(6kbp)(20ng /μl)
      1.25μl正向引物(100 ng /μl或10μM),各突变位点为
      1.25μl反向引物(100 ng /μl或10μM),各自的突变位点为
      5μldNTP-Mix(2.5mM) 5μlPfu-Ultra缓冲液
      1μlPfu-Ultra 2.5U /μl
      35.25μl水
      PCR程序:

      温度
      时间

      95°C
      30秒

      95°C
      30秒
      18个周期
      55°C
      1分钟
      72℃
      1分钟/kbp质粒


    2. 最后,反应补充有1/10体积的10x DpnI缓冲液和10U DpnI。 将反应在37℃下保温1小时
    3. 然后将1.25μl的该混合物转化到超感受态E中。 大肠杆菌DH5α
    4. 序列完整性必须通过顺序来证明。
    5. 为了表达目的,将具有ClcF或其突变体的密码子序列的质粒在E中转化。 大肠杆菌 BL21(DE3)-CP-RIL
    6.  对于大规模蛋白质生产,用来自转化平板的菌落建立过夜培养物。 将培养物在37℃和250rpm下在LB培养基中孵育16小时
    7. 然后,将1L LB培养基以1/100的比例接种过夜培养物,并进一步在37℃和250rpm下孵育。
    8. 如果培养物达到0.6(约2-2.5小时)的光密度(OD 600nm),通过加入1M IPTG储备溶液至终浓度为1mM诱导蛋白质产生。 将培养物在30℃下进一步温育4小时。
    9. 4小时诱导后,在4℃下以4500×g收集细胞25分钟。
    10. 将所得的沉淀物重悬浮在 20ml洗涤缓冲液,并再次在4℃下以4500×g离心45分钟。
    11. 将所得沉淀储存在-20℃直至进一步使用
  2. 净化
    为了得到足够纯度的蛋白质≤95%(由SDS-凝胶估计),需要三步策略,包括热沉淀和两步柱层析。
    1. 将来自1升培养物体积的细胞沉淀重悬于25ml裂解缓冲液中。 加入2M MgCl 2储备溶液至终浓度为3mM,并将0.5U DNase用于消化DNA。
    2. 使用预冷的French Press在1,500Psi下破碎细胞。 将细胞通过French Press三次,以确保完全裂解。
    3. 通过在50,000×g离心1小时除去细胞碎片。
    4. 将上清液在水浴中加热至65℃10分钟,然后在冰上冷却
    5. 通过在50,000×g离心1小时除去变性蛋白
    6. 将所得上清液(大约25ml)施加在用IEX-A缓冲液预平衡的Q-琼脂糖上。
    7. 将蛋白质施加在柱上,并收集含有ClcF的流过液(约30ml)。
    8. 将固体硫酸铵(AMS)加入至前一步骤的流出物(约6g)的最终浓度为1.6M。
    9. 将所得溶液施加到用HIC缓冲液预平衡的苯基琼脂糖上。使用Aekta色谱系统,用20个柱体积的线性梯度洗脱蛋白质至0M AMS。通过如在下面的论文中所述的活性测定来鉴定含ClcF的级分(Roth等人,2013)。
    10. 合并含ClcF的级分,并在4℃下用250倍蛋白体积的25mM Tris(pH7.5)充分透析。 12小时后更换透析缓冲液一次。
    11. 在预结晶之前,用具有30,000kDa的MWCO的VivaSpin浓缩器以4,000×g将蛋白质浓缩至10mg/ml(使用Bradford测定法测定)。将蛋白质的等分试样直接冷冻在-80℃直至进一步使用
  3. 结晶
    1. 基于商业或发布的筛选(例如稀疏矩阵,网格筛选)的配方,制备所有必需的缓冲溶液作为在相关pH下浓度为1M的储备溶液[Jancarik和Kim, 1991; Brzozowski和Walton,2001; Hampton Research(见参考文献5); Jena Bioscience(参见参考文献6)]。
    2. 所有盐溶液和沉淀剂溶液制备为尽可能最高浓度的储备溶液,如果适用,进行过滤。对于ClcF,产生具有pH 6.0和7.0的间隔为0.2pH单位的1M Bis-Tris溶液(用NaOH/HCl调节pH)。此外,需要2M MgCl 2溶液和50%(w/v)PEG 3350的溶液。所有溶液用双蒸水制备。
    3. 通过将预先制备的合适的储备溶液在Eppendorf管中混合来产生各个筛网的条件。
    4. 在Greiner Crystalquick 96孔结晶板中通过具有90μl储液器的移液机器人设置屏幕。
    5. 通过分别将0.2μl储液溶液和0.2μl蛋白溶液一起吸取来设置滴液。不需要进一步混合。然后将板在19℃下储存。然后定期检查板(在第一周每天,之后每周)以形成晶体。晶体通常出现在一天和长达3个月之间,但偶尔甚至更长的时间可能需要获得晶体。如果鉴定到有希望的条件(晶体,微晶或结晶沉淀),则通常通过在±1.5pH单位的范围内改变初始条件附近的pH,在悬滴24孔形式中设置细筛,范围(图1)。


      图1.二维24井网格屏幕的典型方案,同时改变pH和沉淀浓度

    6. 如所述制备贮存器,通过在24孔Linbro结晶板中将1μl蛋白质与1μl贮存器在500μl贮存器中混合,以2μl的总滴体积设置液滴。
    7. 对于ClcF的正交晶体,在所有条件下具有0.4M MgCl 2的恒定浓度的筛,如图2所示。


      图2.正交ClcF晶体,同时改变pH和沉淀剂浓度的典型屏幕。在所有条件下,MgCl 2终浓度为0.4M。

    8. 对于复合物,将蛋白质与10mM底物(5-氯葡萄糖酸内酯)预混合,所述底物以1M的浓度溶解在pH 7的水中(用NaHCO 3调节)。在结晶之前不需要延长的孵育期。结晶板的设置如上所述进行

      图3.在蛋白质沉淀中作为进一步细筛选的起始点(左)和ClcF的典型单晶(右)的前体结晶材料。更多图片可以在Hampton Research网站上找到(参见参考文献5 )。

  4. 冷冻保护
    1. 选择具有良好晶体的条件,并且用补充有25%乙二醇的2%升高的沉淀剂浓度制备储存溶液。
    2. 随后少量地,将0.1至0.2μl的冷冻溶液加入到滴液中,并且滴液与冷冻溶液缓慢地交换。
    3. 在复合物的情况下,冷冻溶液含有终浓度为25%的沉淀物加上浓度为10mM的配体。 不需要额外的乙二醇。
    4. 最后,用尼龙环捕获晶体并倒入液氮中。

食谱

  1. 裂解缓冲液/离子交换(IEX)缓冲液A /疏水相互作用层析(HIC)缓冲液A
    25mM Tris-HCl(pH7.5)
  2. HIC高盐缓冲液
    25mM Tris-HCl(pH7.5)(加入所有成分后的最终pH) 1.6M硫酸铵
  3. 洗涤缓冲液
    50mM Tris(pH7.0)
    150mM NaCl
  4. 结晶缓冲液
    用HCl或NaOH调节pH

致谢

作者使用由所述研究人员开发的以下标准方案。如Bradford(1976)中所述的Bradford测定法。如Laemmli(1970)中所述的SDS-凝胶电泳。用于培养基和标准缓冲液的标准配方来自:"Molecular Cloning。"Green,M.R。和Sambrook,J.Cold Spring Harbor Laboratory Press。

参考文献

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引用:Roth, C., Gröning, J. A., Kaschabek, S. R., Schlömann, M. and Sträter, N. (2014). Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP. Bio-protocol 4(8): e1107. DOI: 10.21769/BioProtoc.1107.
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