生产，结晶和结构测定<em>℃。难辨</em>通过Microseeding和锌SAD PPEP-1
Summary
Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.
Abstract
New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.
Introduction
难辨梭状芽孢杆菌是院内抗生素相关性腹泻感染1的主要原因之一。这种革兰氏阳性厌氧细菌通过其孢子形式通过粪 – 口途径传播。在过去的十年，新的'流行''或''超毒'株（ 如 BI / NAP1 / 027）造成的在北美和欧洲的2个新的感染和死亡率急剧增加。 艰难梭菌病机相关性（CDAD）是一种威胁生命结肠炎的高死亡率3。症状的范围从腹泻4至伪膜性肠炎5和经常致命的中毒性巨结肠6。
CDAD治疗是困难的，因为该毒株是耐多药，复发率高7。此刻疗法包括抗生素甲硝唑，fidaxomicin或万古霉素，或者在repetitively复发病例粪便菌群移植。新的治疗策略，迫切需要8。一些进展记录为治疗性单克隆抗体Bezlotoxumab，瞄准艰难梭菌毒素B 9，日前顺利通过III期临床试验，并申请了与FDA和EMA的批准。此外，新的抗生素被此刻测试在临床试验10的不同阶段。
要制定有效的治疗新的治疗靶点必须进行标识。最近发现的艰难梭菌蛋白酶脯氨酸脯氨酸内肽酶-1（PPEP-1; CD2830 / Zmp1; EC 3.4.24.89）是这样一种有前途的靶，如在敲除菌株缺乏PPEP-1的降低的C毒力。难辨体内 11。 PPEP-1是分泌金属蛋白酶12,13在它们的C-末端裂解2 梭状粘附13从而释放粘合杆菌IA从人体肠道上皮细胞。因此，参与维持艰难梭菌的固着和能动表型之间的平衡。为了开发选择性抑制剂对PPEP-1，了解它是如何认识它的底物的三维结构的深入了解是必不可少的。我们已经解决PPEP-1单独和复合物的第一晶体结构的衬底的肽14。 PPEP-1是第一个已知的蛋白酶的两个脯氨酸残基的15之间选择性裂解肽键。其结合在双扭结方式在基片和通过位于在S环覆盖蛋白酶活性位点14个残基的延伸脂族-芳族的网络稳定它。此底物结合模式是唯一的PPEP-1和人蛋白酶迄今未找到。这使得它有前途的药物靶标，以及脱靶酶抑制剂不太可能的影响。
为了发展和屏幕选择PPEP-1 INHibitors在将来需要大量的纯净，单分散PPEP-1蛋白。此外，为了确定与PPEP-1第一抑制剂，共晶体结构的结合将要确定的模式。在我们的手中PPEP-1不断推出共生晶体。因此，我们已开发出一种优化过程，以产生PPEP-1的单衍射质量的晶体。在这个协议中，我们详细描述了PPEP-1 14的生产，纯化，结晶和结构的解决方案。我们在缺乏分泌信号序列，亲和层析和大小排阻色谱法除去纯化标记的一个PPEP-1变体的大肠杆菌用胞内表达，随后通过锌单波长异常色散microseeding 16成优化屏幕和结构确定（锌- SAD）17。这个协议可以适于生产和结构确定的其他蛋白质（ 例如</ em>的金属蛋白酶），特别是用于生产共生晶体蛋白质。根据要求，构建体的质粒DNA（质粒pET28a-NHIS-rPPEP-1）和衍射数据可以提供用于教育目的。
Protocol
Representative Results
Discussion
X射线晶体学仍然是确定的蛋白质28三维近原子分辨率的结构最快，最准确的方法。但是，它需要秩序井然单晶的生长。这些往往难以得到与结晶状态是人为的。然而，通过X射线晶体与用其他方法，测定确定蛋白质结构的比较特别NMR，通常显示出非常良好的一致性。在PPEP-1的情况下，一个NMR结构最近发表29示出良好的协议与我们的晶体结构14，包括S-环路的流动性。
<p cla…Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢员工同步数据采集期间在瑞士光源，保罗谢勒研究所，菲利根，瑞士支持束线X06DA。我们感谢莫妮卡Gompert优秀的技术支持。该项目由科隆大学，并授予由德国研究委员会INST 216 / 682-1 FUGG支持。从国际研究生院在发展健康和疾病给CP一个博士研究生奖学金是公认的。导致这些结果的研究号赠款协议283570（BioStruct-X）下收到的资金来自欧盟第七框架计划（FP7 / 2007-2013）。
Materials
| Genes / Vectors / cell strains | |||
| pET28a vector | Merck-Millipore | 69864 | Thrombin cleavable N-terminal His-tag |
| E. coli strain BL21 (DE3) Star | ThermoFisher Scientific | C601003 | RNase H deficient |
| Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300) | Geneart (Thermo Fisher Scientific) | custom | amino acids 27-220 |
| Name | Company | Catalog Number | Comments |
| Chemicals | |||
| Yeast extract | any | ||
| Tryptone | any | ||
| Antifoam B | Sigma-Aldrich | A5757 | aqueous-silicone emulsion |
| Agar | any | ||
| Kanamycin | any | ||
| IPTG | AppliChem | A1008 | |
| Tris-HCl | AppliChem | A1087 | Buffer grade |
| NaCl | any | Buffer grade | |
| DNaseI | AppliChem | A3778 | |
| Imidazole | AppliChem | A1073 | Buffer grade |
| Thrombin | Sigma-Aldrich | T4648 | |
| Ammonium phosphate dibasic | Sigma-Aldrich | 215996 | |
| Glycerol 100% | any | purest grade | |
| Sucrose | Sigma-Aldrich | 84097 | |
| Liquid nitrogen | any | for storage and cryocooling of crystals | |
| Name | Company | Catalog Number | Comments |
| Equipment (general) | |||
| Shaking incubator | any | providing temperatures of 20 °C – 37 °C | |
| Glassware | any | baffled Erlenmeyer flasks (50 ml – 2.8L) | |
| Centrifuge for large culture volumes | any | centrifuge for processing volumes up to 12 L | |
| Sonicator Vibra-Cell VCX500 | Sonics | SO-VCX500 | or any other sonicator / cell disruptor |
| Ultracentrifuge | any | centrifuge providing speeds up to 150.000 x g | |
| NiNTA Superflow resin | Qiagen | ||
| Empty Glass Econo-Column | Bio-Rad | 7371007 | or any other empty glass or plastic column |
| Size exclusion chromatography column HiLoad Superdex 200 16/600 | GE Healthcare | 28989335 | |
| Chromatography system Äkta Purifier | GE Healthcare | 28406264 | or any other chromatography system |
| Dialysis tubing Spectra/Por 3 | Spectrum Labs | 132724 | |
| Dialysis tubing closures | Spectrum Labs | 132738 | |
| Ultrafiltration units (concentrators) 10.000 NWCO | any | ||
| UV-Vis spectrophotometer | any | ||
| Name | Company | Catalog Number | Comments |
| Equipment (crystallography) | |||
| Low volume pipette 0.1-10 µl | any | ||
| Positive displacement pipette Microman M10 | Gilson | F148501 | |
| Crystallization robot | any | ||
| 96-well crystallization plates TTP IQ with three protein wells | TTP | 4150-05810 | or any other 96-well crystallization plate |
| 24-well CombiClover Junior Plate | Jena Bioscience | EB-CJR | |
| Crystal Clear Sealing Tape | Hampton Research | HR3-511 | |
| Siliconized Glass Cover Slides | Hampton Research | HR3-225 | |
| Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal | Hampton Research | diverse | |
| Commercial crystallization screens: Wizard, PACT++, JCSG++ | Jena Bioscience | diverse | |
| JBS Beads-for-Seeds | Jena Bioscience | CO-501 | |
| CrystalCap SPINE HT (nylon loops) | Hampton Research | diverse | loop sizes 0.025 mm – 0.5 mm |
| CrystalCap Vial | Hampton Research | HR4-904 | |
| Cryogenic Foam Dewar 800 ml | Hampton Research | HR4-673 | |
| Cryogenic Foam Dewar 2L | Hampton Research | HR4-675 | |
| Vial Clamp, Straight | Hampton Research | HR4-670 | |
| CrystalWand Magnetic, Straight | Hampton Research | HR4-729 | |
| CryoCane 6 Vial Holder | Hampton Research | HR4-711 | |
| CryoSleeve | Hampton Research | HR4-708 | |
| CryoCane Color Coder – White | Hampton Research | HR4-713 | |
| Scalpel | any | ||
| Straight microforcep | any | for manipulation of sealing tape. etc. | |
| Acupuncture needle | any | e.g. from a pharmacy | |
| Stereo microscope | any | for inspection of crystallization plates and crystal mounting, magnification up to 160X |
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