Name: production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad
Uploaded: 2016-12-30T15:00:00.000+00:00
Duration: 13 min 34 s
Description: Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

우리는 싱크로트론 데이터 수집시 지원을위한 스위스 광원, 폴 Scherrer의-연구소, Villigen, 스위스의 빔라인의 X06DA에서 직원을 감사드립니다. 우리는 우수한 기술 지원 모니카 Gompert에 감사하고 있습니다. 이 프로젝트는 쾰른 대학에서 지원과 독일 연구 협의회에서 INST 216 / 682-1 FUGG을 부여했다. CP에 개발 건강과 질병의 국제 대학원에서 박사 교제는 인정된다. 이러한 결과로 이어지는 연구는 보조금 계약 번호 283570 (BioStruct-X)에서 유럽 공동체의 일곱 번째 프레임 워크 프로그램 (FP7 / 2007-2013)에서 자금을 받았다.

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The authors have nothing to disclose.

X 선 결정학은 여전히 단백질 (28)의 입체에 가까운 해상도 원자 구조를 결정하기위한 가장 빠르고 가장 정확한 방법이다. 그러나, 잘 정렬 된 단결정의 성장을 필요로한다. 이들은 종종 얻을 수 어렵고, 결정 상태는 인공이다. 그러나, 다른 방법에 의해 결정된 것과 X 선 결정학에 의해 결정된 구조 단백질의 비교 특히 NMR, 일반적으로 매우 잘 일치 함을 나타낸다. PPEP-1의 경우에, NMR 구조는 ...

클로스 트리 디움 디피는 원내 항생제 관련 설사 감염 1의 주요 원인 중 하나입니다. 이 그람 양성 혐기성 세균은 분변 - 경구 경로를 통해 그 포자 양식을 통해 전송된다. 지난 10 년, 새로운 &#39;전염병&#39; &#39;또는&#39; &#39;hypervirulent&#39; &#39;균주 (/ 027 예를 들어, BI / NAP1)는 북미와 유럽이 새로운 감염과 사망률의 급격한 증가가 발생했습니다. C. 디피 -associated 질환 (CDAD)는 높은 사망률 3 생명을 위협하는 대장 염증이다. 증상은 설사 4에서 위 막성 대장염 (5)와 종종 치명적 독성 거대 결장 (6)에 이르기까지 다양합니다. 유독 균주 다제 내성 및 재발 7 높다으로 CDAD 치료가 어렵다. 현재 치료법은 항생제 메트로니다졸, fidaxomicin 또는 반코마이신 또는 repetiti에 포함에서vely 재발하는 경우 분변 미생물 이식. 새로운 치료 전략이 시급히 8 필요하다. 일부 진보가 C. 디피 독소 B (9)을 대상으로, 치료 단클론 항체 Bezlotoxumab로 기록, 최근 성공적으로 임상 3 상을 통과하고 FDA 및 EMA에 승인을 신청했다. 또한, 새로운 항생제 임상 시험 (10)의 다른 단계에서 순간에서 시험되고있다. 새로운 치료 표적을 식별해야 효과적인 치료를 개발합니다. (; CD2830 / Zmp1, PPEP-1 EC 3.4.24.89)가 최근 C. 디피 단백질 분해 효소 프롤린 - 프롤린 엔도 펩 티다 제-1 발견 노크 아웃 변형에 PPEP-1의 부족 등 유망한 대상이되고, C의 독성을 감소 . 생체 내 11 디피. PPEP-1은 C 말단에 두 C. 디피의 adhesins를 절단 분비 메탈로 12, 13 (13), 따라서 부착 bacter를 해제인간의 장내 상피 세포에서 아이오와. 따라서, C. 디피의 정착과 운동성 표현형 사이의 균형을 유지에 관여한다. PPEP-1에 대해 선택적 저해제를 개발하고 그 기판의 입체 구조의 자세한 지식이 필요 불가결 인식하는 방식을 이해하는 것이. 우리는 기질 펩티드 14 PPEP-1 단독 컴플렉스의 제 결정 구조를 해결했다. PPEP-1은 제 1 공지 프로테아제 두 개의 프롤린 잔기 (15) 사이를 선택적으로 절단하여 펩티드 결합. 이것은 이중 꼬임 방식으로 기판에 결합하고, 프로테아제 활성 부위 (14)를 덮는 S 루프에있는 잔기 연장 된 지방족 - 방향족 네트워크를 통해 안정화. 이 기판 결합 모드는 PPEP-1에 고유하며 현재까지 인간의 단백질 분해 효소에서 찾을 수 없습니다. 이것은 유망한 약물 표적하게, 매우 가능성 억제제의 오프 대상 효과. 화면 선택 PPEP-1 INH 개발하려면ibitors은 향후 순수한 단 분산 PPEP-1 단백질의 다량이 필요하다. 또한, 결정되어야 할 것이다 PPEP-1 제 억제제, 공동 결정 구조의 결합 모드를 결정한다. 우리의 손에 PPEP-1은 지속적으로 (intergrown)의 결정을 생성합니다. 따라서 우리는 PPEP-1의 단일 회절 품질의 결정을 생성하는 최적화 과정을 개발 하였다. 이 프로토콜에서 우리는 구체적으로 PPEP-1 (14)의 생산, 정제, 결정화 및 구조 솔루션을 설명합니다. 우리는 분비 신호 서열, 정제 태그의 제거와 친 화성 크로마토 그래피 및 크기 배제 크로마토 그래피 부족한 PPEP-1 변이체의 대장균 세포 내 발현을 사용하여, 아연의 단일 파장 이상 분산을 통해 최적화 화면 구조 결정에 16 microseeding 이어 (아연 SAD) 17. 이 프로토콜은 (다른 단백질의 제조 및 구조 결정에 적용 할 수있다 예를 들어, &lt;/ EM&gt; 메탈로) 및 (intergrown)의 결정을 생산하는 단백질 관한 것이다. 요청시 구조물의 플라스미드 DNA는 (는 pET28a-는 NHIS-rPPEP-1)의 회절 데이터는 교육 목적으로 제공 될 수있다.

<table><tbody><tr><td>&lt;strong&gt;Genes / Vectors / cell strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET28a vector</td><td>Merck-Millipore</td><td>69864</td><td>Thrombin cleavable N-terminal His-tag</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; strain BL21 (DE3) Star</td><td>ThermoFisher Scientific</td><td>C601003</td><td>RNase H deficient</td></tr><tr><td>Codon-optimized gene (for &lt;em&gt;E. coli&lt;/em&gt;) of PPEP-1 (CD630_28300)</td><td>Geneart (Thermo Fisher Scientific)</td><td>custom</td><td>amino acids 27-220</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Yeast extract</td><td>any</td><td></td><td></td></tr><tr><td>Tryptone</td><td>any</td><td></td><td></td></tr><tr><td>Antifoam B</td><td>Sigma-Aldrich</td><td>A5757</td><td>aqueous-silicone emulsion</td></tr><tr><td>Agar</td><td>any</td><td></td><td></td></tr><tr><td>Kanamycin</td><td>any</td><td></td><td></td></tr><tr><td>IPTG</td><td>AppliChem</td><td>A1008</td><td></td></tr><tr><td>Tris-HCl</td><td>AppliChem</td><td>A1087</td><td>Buffer grade</td></tr><tr><td>NaCl</td><td>any</td><td></td><td>Buffer grade</td></tr><tr><td>DNaseI</td><td>AppliChem</td><td>A3778</td><td></td></tr><tr><td>Imidazole</td><td>AppliChem</td><td>A1073</td><td>Buffer grade</td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T4648</td><td></td></tr><tr><td>Ammonium phosphate dibasic</td><td>Sigma-Aldrich</td><td>215996</td><td></td></tr><tr><td>Glycerol 100%</td><td>any</td><td></td><td>purest grade</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>Liquid nitrogen</td><td>any</td><td></td><td>for storage and cryocooling of crystals</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (general)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shaking incubator</td><td>any</td><td></td><td>providing temperatures of 20 &amp;deg;C - 37 &amp;deg;C</td></tr><tr><td>Glassware</td><td>any</td><td></td><td>baffled Erlenmeyer flasks (50 ml - 2.8 L)</td></tr><tr><td>Centrifuge for large culture volumes</td><td>any</td><td></td><td>centrifuge for processing volumes up to 12 L</td></tr><tr><td>Sonicator Vibra-Cell VCX500</td><td>Sonics</td><td>SO-VCX500</td><td>or any other sonicator / cell disruptor</td></tr><tr><td>Ultracentrifuge</td><td>any</td><td></td><td>centrifuge providing speeds up to 150,000 x g</td></tr><tr><td>NiNTA Superflow resin</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Empty Glass Econo-Column</td><td>Bio-Rad</td><td>7371007</td><td>or any other empty glass or plastic column</td></tr><tr><td>Size exclusion chromatography column HiLoad Superdex 200 16/600</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>Chromatography system &amp;Auml;kta Purifier</td><td>GE Healthcare</td><td>28406264</td><td>or any other chromatography system</td></tr><tr><td>Dialysis tubing Spectra/Por 3</td><td>Spectrum Labs</td><td>132724</td><td></td></tr><tr><td>Dialysis tubing closures</td><td>Spectrum Labs</td><td>132738</td><td></td></tr><tr><td>Ultrafiltration units (concentrators) 10,000 NWCO</td><td>any</td><td></td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>any</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (crystallography)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Low volume pipette 0.1-10 &amp;micro;l</td><td>any</td><td></td><td></td></tr><tr><td>Positive displacement pipette Microman M10</td><td>Gilson</td><td>F148501</td><td></td></tr><tr><td>Crystallization robot</td><td>any</td><td></td><td></td></tr><tr><td>96-well crystallization plates TTP IQ with three protein wells</td><td>TTP</td><td>4150-05810</td><td>or any other 96-well crystallization plate&amp;nbsp;</td></tr><tr><td>24-well CombiClover Junior Plate</td><td>Jena Bioscience</td><td>EB-CJR</td><td></td></tr><tr><td>Crystal Clear Sealing Tape</td><td>Hampton Research</td><td>HR3-511</td><td></td></tr><tr><td>Siliconized Glass Cover Slides</td><td>Hampton Research</td><td>HR3-225</td><td></td></tr><tr><td>Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td><td>Hampton Research</td><td>diverse</td><td></td></tr><tr><td>Commercial crystallization screens: Wizard, PACT++, JCSG++</td><td>Jena Bioscience</td><td>diverse</td><td></td></tr><tr><td>JBS Beads-for-Seeds</td><td>Jena Bioscience</td><td>CO-501</td><td></td></tr><tr><td>CrystalCap SPINE HT (nylon loops)</td><td>Hampton Research</td><td>diverse</td><td>loop sizes 0.025 mm - 0.5 mm</td></tr><tr><td>CrystalCap Vial</td><td>Hampton Research</td><td>HR4-904</td><td></td></tr><tr><td>Cryogenic Foam Dewar 800 ml</td><td>Hampton Research</td><td>HR4-673</td><td></td></tr><tr><td>Cryogenic Foam Dewar 2 L</td><td>Hampton Research</td><td>HR4-675</td><td></td></tr><tr><td>Vial Clamp, Straight</td><td>Hampton Research</td><td>HR4-670</td><td></td></tr><tr><td>CrystalWand Magnetic, Straight</td><td>Hampton Research</td><td>HR4-729</td><td></td></tr><tr><td>CryoCane 6 Vial Holder</td><td>Hampton Research</td><td>HR4-711</td><td></td></tr><tr><td>CryoSleeve</td><td>Hampton Research</td><td>HR4-708</td><td></td></tr><tr><td>CryoCane Color Coder - White</td><td>Hampton Research</td><td>HR4-713</td><td></td></tr><tr><td>Scalpel</td><td>any</td><td></td><td></td></tr><tr><td>Straight microforcep</td><td>any</td><td></td><td>for manipulation of sealing tape. &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Acupuncture needle</td><td>any</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; from a pharmacy</td></tr><tr><td>Stereo microscope</td><td>any</td><td></td><td>for inspection of crystallization plates and crystal mounting, magnification up to 160X</td></tr></tbody></table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

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.

rPPEP-1은 대장균 BL21 (DE3) 스타 (도 1C)에서 가장 높은 수율로 여러 대장균 균주에서 과발현된다. 제 NiNTA 친 화성 크로마토 그래피 단계 후 6xHis 태그가 성공적으로 단백질의 대부분으로부터 절단 될 수 있으며, 상기 제 NiNTA 단계에서 소화되지 않은 단백질은 완전히 트롬빈 분해 단백질 (도 1D)로부터 분리 될 수있다. 가끔 대부분의 아마 ...

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

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.

생산, 결정화 및 구조 결정의<em&gt; C. 남과 어울리지 않는</em&gt; PPEP-1 Microseeding 및 아연 SAD를 통해

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 ...

production-crystallization-structure-determination-c-difficile-ppep-1

Research

JoVE Journal

Biochemistry

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

11.5K 조회수.  University of Cologne. 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. 

비디오: Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

구조 연구 생산 이황화 - 안정화 Transmembrane의 펩타이드 단지

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

연구

화학

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

높은 명령으로 Nanosheets에 Peptoid 폴리머 및 자기 조립의 고체 상 합성 Submonomer

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

생체공학

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

탄수화물 전송 기질 바인딩 단백질 SP0092의 생 화 학적 및 구조적 특성

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

생화학

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

인플루엔자 중합 효소의 유전자에 대한 구조 결정 PB2 소단위를위한 멀티 타겟 병렬 처리 방식

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

면역학 및 감염병학

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

배양 및 유지<em&gt; 클로 스트 리듐 디피</em&gt; 혐기성 환경에서

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

열토가 마리티마 M42 아미노펩티다제 TmPep1050의 올리고메릭 상태 전환을 연구하는 엑스레이 결정학

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

연속 결정학 실험을 위한 Endothiapepsin 결정의 성장 최적화

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

다목적 미세 유체 칩의 직렬 결정학에 의한 효소의 결정화 및 구조적 결정: 기질 복합체

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

세균 포자 발아 동안 코어 텍스 조각 검출

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

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