Name: production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad
Uploaded: 2016-12-30T15:00:00
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.

1. 복제 및 디자인을 구축 <ol><li> 신호 펩타이드없이 C. 디피 PPEP-1 (대장균) 코돈 최적화 된 시퀀스를 복제 [아미노산 27-220, 이하 재조합 PPEP -1- 이름 (rPPEP-1) 11]을 Nde I을 사용는 pET28a 벡터에 및을 XhoI 제한 부위 3&#39;- 말단 (생성 된 벡터는 pET28a-NHIS-rPPEP-1)에서 종료 코돈과 (도 1). 이는 정제 과정 태그를 제거 가능 트롬빈 절단 부위 (도 1)와...

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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 border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Genes / Vectors / cell strains</td>
			<td style="width:83px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET28a vector</td>
			<td>Merck-Millipore</td>
			<td>69864</td>
			<td>Thrombin cleavable N-terminal His-tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli strain BL21 (DE3) Star</td>
			<td>ThermoFisher Scientific</td>
			<td>C601003</td>
			<td>RNase H deficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300)</td>
			<td>Geneart (Thermo Fisher Scientific)</td>
			<td>custom</td>
			<td>amino acids 27-220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam B</td>
			<td>Sigma-Aldrich</td>
			<td>A5757</td>
			<td>aqueous-silicone emulsion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>AppliChem</td>
			<td>A1008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>any</td>
			<td></td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNaseI</td>
			<td>AppliChem</td>
			<td>A3778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>AppliChem</td>
			<td>A1073</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>215996</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>any</td>
			<td></td>
			<td>purest grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>any</td>
			<td></td>
			<td>for storage and cryocooling of crystals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (general)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking incubator</td>
			<td>any</td>
			<td></td>
			<td>providing temperatures of 20 &#176;C - 37 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glassware</td>
			<td>any</td>
			<td></td>
			<td>baffled Erlenmeyer flasks (50 ml - 2.8L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge for large culture volumes</td>
			<td>any</td>
			<td></td>
			<td>centrifuge for processing volumes up to 12 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator Vibra-Cell VCX500</td>
			<td>Sonics</td>
			<td>SO-VCX500</td>
			<td>or any other sonicator / cell disruptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifuge</td>
			<td>any</td>
			<td></td>
			<td>centrifuge providing speeds up to 150.000 x g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NiNTA Superflow resin</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Empty Glass Econo-Column</td>
			<td>Bio-Rad</td>
			<td>7371007</td>
			<td>or any other empty glass or plastic column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Size exclusion chromatography column HiLoad Superdex 200 16/600</td>
			<td>GE Healthcare</td>
			<td style="width:136px;">28989335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography system &#196;kta Purifier</td>
			<td>GE Healthcare</td>
			<td>28406264</td>
			<td>or any other chromatography system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing Spectra/Por 3</td>
			<td>Spectrum Labs</td>
			<td>132724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing closures</td>
			<td>Spectrum Labs</td>
			<td>132738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafiltration units (concentrators) 10.000 NWCO</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis spectrophotometer</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (crystallography)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low volume pipette 0.1-10 &#181;l</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Positive displacement pipette Microman M10</td>
			<td>Gilson</td>
			<td>F148501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystallization robot</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well crystallization plates TTP IQ with three protein wells</td>
			<td>TTP</td>
			<td style="width:136px;">4150-05810</td>
			<td>or any other 96-well crystallization plate&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well CombiClover Junior Plate</td>
			<td>Jena Bioscience</td>
			<td>EB-CJR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystal Clear Sealing Tape</td>
			<td>Hampton Research</td>
			<td>HR3-511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Siliconized Glass Cover Slides</td>
			<td>Hampton Research</td>
			<td>HR3-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: Wizard, PACT++, JCSG++</td>
			<td>Jena Bioscience</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JBS Beads-for-Seeds</td>
			<td>Jena Bioscience</td>
			<td>CO-501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">CrystalCap Vial</td>
			<td>Hampton Research</td>
			<td>HR4-904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 800 ml</td>
			<td>Hampton Research</td>
			<td>HR4-673</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 2L</td>
			<td>Hampton Research</td>
			<td>HR4-675</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vial Clamp, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalWand Magnetic, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane 6 Vial Holder</td>
			<td>Hampton Research</td>
			<td>HR4-711</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoSleeve</td>
			<td>Hampton Research</td>
			<td>HR4-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane Color Coder - White</td>
			<td>Hampton Research</td>
			<td>HR4-713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight microforcep</td>
			<td>any</td>
			<td></td>
			<td>for manipulation of sealing tape. etc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acupuncture needle</td>
			<td>any</td>
			<td></td>
			<td>e.g. from a pharmacy</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Stereo microscope</td>
			<td>any</td>
			<td></td>
			<td style="width:191px;">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 ...

Research

JoVE Journal

Biochemistry

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

효능 판별 분석에서 펩타이드 작용제의 직렬 희석에 대한 분배 자동 음향

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