이 작품은 이스라엘 과학 재단 재단 연구 센터 (I-CORE)의 구조 세포 생물학 (1775/12)과 이스라엘 과학 재단 보조금 (1721/16과 2338/16) (YH)과 825/14 (DK ). DK에 대한 Fields Estate Foundation의 지원은 높이 평가됩니다. 이 연구는 Ilan Edri와 Michal Goldenberg가 Tel Aviv University의 Sackler 의학부의 MD 논문 요구 사항을 부분적으로 이행함으로써 수행되었습니다.

1 . E. coli 에서의 과발현을위한 cis -PT의 클로닝 <ol><li> 109aa thioredoxin (TRX) 단백질 17 과 융합 된 단백질 서열의 클로닝 및 고수준 발현을 위해 설계된 pET-32b 발현 벡터 및 대장균 코돈 최적화 된 전장 cis -PT 18 코딩 서열을 수득한다. </li><li> TEV 프로테아제 짧은가요 성 링커 하였다 (담배 식각 바이러스 단백질 분해 효소) 절단 부위 ( &quot;/&quot;는 절단 점을 나타내는 ENLYFQ / G) 9 갖도록주의 시스의 상류 (SGSGSG을 절단 부위의 접근성을 향상시키기) -PT 서열 ( 도 1A ). 또한, 복제에 적합한 5 &#39;및 3&#39;제한 사이트를 추가하십시오. </li><li> 표준 ligation 기술을 사용하여 합성 DNA 구조를 pET-32b 벡터에 클로닝하십시오 10 . </li></ol> 2. 지나치게 높은E. coli 에서의 인간 DHDDS의 절단 <ol><li> 제조사의 지시에 따라 TRX- 융합 DHDDS 구조체로 대장균 T7을 형질 전환시킨다. </li><li> 암피실린 선택 조건 (200 MG / L)하에 LB - 한천에 대한 제조 업체의 지침에 따라 형질 전환 세포를 플레이트. </li><li> 100 밀리그램 / L의 암피실린과 250 ML 플라스크 스타터 문화의 LB 매체 100 ML에 선택한 식민지를 접종하고 200 rpm으로 일정한 흔들림과 37 ° C에서 하룻밤 incubate. </li><li> 이 배양액 80 mL를 2 x YT 배지 (16 g / L 트립 톤, 10 g / L 효모 추출물, 5 g / L NaCl) 1.5 L가 각각 함유 된 2 개의 5 L 플라스크 (플라스크 당 40 mL) L 암피실린. </li><li> OD 600 nm = 0.5에 도달 할 때까지 180 rpm으로 일정하게 흔들어 주면서 37 ° C에서 배양합니다. </li><li> 온도를 16 ° C로 낮추고 0.5 mM 이소 프로필 β-D-1- 티오 갈 락토 피 라노 시드 (IPTG)를 첨가하여 세포를 유도하십시오. 범죄자단백질 발현을 위해 동일한 조건 하에서 16-20 시간 항온 처리한다. </li><li> 원심 분리 (10,000 x g 10 분)에 의해 세포를 수확. 참고 : 여기서 프로토콜을 일시 중지 할 수 있습니다. 세포 펠릿은 정화 때까지 -80 ° C에서 보관해야합니다. </li></ol> 삼 . 인간 DHDDS의 정제 <ol><li> 버퍼 A (25 MM 트리스 - HCL, 산도 7.5, 150 MM NaCl, 0.1 % 트리톤 X - 100, 10 MM β - mercaptoethanol), 1 μg / ML DNase I, 그리고 프로 테아 제 억제제 혼합물에있는 세포를 Resuspend. </li><li> 유리 - 테플론 균질기를 사용하여 세포를 균질화하십시오. </li><li> 15,000 PSI 11 - 12,000 마이크로 플루 또는 상응하여 세포를 방해. </li><li> 4 ° C에서 45 분 동안 40,000 xg에서 세포 용 해물을 원심 분리하십시오. 가용성 분획을 포함하는 상청액을 회수한다. </li><li> 상층 액을 5 - 10 mL 코발트 고정화 금속 친 화성 크로마토 그래피 (Co 2+ -IMAC) 12 컬럼에 넣고 10이후에 완충액 A 및 10 mM 이미 다졸로 철저히 세척하여 비특이적 단백질 결합을 감소시켰다. </li><li> 과잉 발현 된 단백질을 완충액 A로 250 mM 이미 다졸로 보충하십시오. </li><li> 완충액 A로 평형화 된 53 mL 예비 탈염 칼럼을 사용하여 이미 다졸을 제거한다. </li><li> 하룻밤 4 ° C에서 6xHis - 태그 TRX 융합 단백질을 제거하기 위해 용출 단백질에 6xHis 태그 TEV 프로 테아 제 (50mg 단백질 당 1mg TEV 프로 테아 제)를 첨가하십시오. </li><li> cleaved 6x His - 태그 TRX 융합 단백질과 TEV 프로 테아 제를 제거하기 위해 5 MM 이미 다졸로 보충 버퍼 A와 함께 평형 Co 2 + -IMAC 컬럼에 절단 단백질을로드합니다. </li><li> 30 kDa 분자량 차단 원심 분리 필터를 사용하여 3 ~ 4 mL로 유출액과 농축 물을 수집합니다. </li><li> 최종 정제를 위해 버퍼 A로 평형화 된 superdex-200 크기 - 배제 크로마토 그래피 컬럼 상에 농축 된 단백질을 로딩한다. 단백질은 이량 체 (~ 76 kDa)로 용리됩니다. </li&gt; <li> 컬럼 분석 곡선과 용출 프로파일을 비교하여 관련 분율을 선택하십시오. </li><li> SDS-PAGE를 사용하여 준비의 순도를 평가하십시오. </li><li> 다운 스트림 실험에 필요한 단백질 농도를 결정하고 사용하기 전까지 액체 질소에서 정화되고 농축 된 단백질의 플래시 동결 분취 량을 측정하고 -80 ° C에서 보관하십시오. </li></ol> 4. 분석적인 크기 배제 크로마토 그래피 (SEC) <ol><li> 동결 융해 사이클을 견딜 수있는 단백질의 능력을 보장하기 위해 동결 단백질의 분량을 해동하고 21,000 x g 에서 10 분간 원심 분리하여 불용성 응집체를 제거합니다. 뜨는을 수집합니다. </li><li> 상 단백질 넣 캅토-200 분석 컬럼 25 mM 트리스 - 염산, pH가 7.5, 150 mM의 NaCl로 미리 평형, 0.02 % 트리톤 X-100, 10 밀리미터 β 머 캅토 에탄올, 초 고성능 액체 크로마토 그래피 시스템 (13)을 사용하여, 14 . </li><li> 트립토판 플루 오레 모니터scence (λ ex = 280 nm, λ em = 350 nm)를 사용하여 단백질의 용출 프로필을 검출합니다. 분자량 평가를위한 올바른 보정 곡선을 가져야합니다. 이러한 곡선은 SEC 컬럼 제조업체 를 통해 구할 수 있습니다. </li></ol> 5. 효소 반응 속도론 - 시간 의존적 활동 15 , 16 , 17 <ol><li> 정제 단백질의 활동을 보장하기 위해 10 μM FPP와 50 μM 14 C - IPP와 정제 DHDDS 5 μM을 섞어 22 - 30 ° C에서 0.5 MM MgCl 2 와 버퍼 A의 반응을 시작합니다. 참고 : 정확한 반응 조건은 DHDDS와는 다른 cis -PT의 준비에 따라 다를 수 있습니다. 또한, DHDDS의 활성도는 온도와 [Mg 2+ ]에 따라 달라집니다. 주의 : 14 C-IPP는 방사성 물질이므로 해당 지역의 방사선 안전 규정에 따라 사용해야합니다. </li&gt; <li> 0, 2, 4 및 6 시간에 반응에서 15 μL 샘플을 철회 후, 15 밀리 버퍼 A 보충 20 MM EDTA (10 MM EDTA의 최종 농도)의 추가로 반응의 즉각적인 급냉 다음. </li><li> H 2 O가 포화 된 1- 부탄올 1 mL를 넣고 충분히 소용돌이 칠하여 반응 생성물을 추출한다. 참고 : 여기서 프로토콜을 중지하고 샘플을 나중에 읽을 수 있도록 보관할 수 있습니다. </li><li> 샘플에 신틸레이션 칵테일을 넣고 신틸레이션 카운터를 사용하여 총 방사능을 나타내는 15 μL의 방사능과 함께 14 C를 포함하는 부탄올 단계의 반응 생성물을 정량하십시오. </li><li> 각 시점에서 0 시간 샘플을 사용하여 측정 된 배경 판독 값을 뺀 후 14 C-IPP의 백분율을 계산합니다. </li><li> 14 개의 이용 비율을 계산하여 각 시점에서의 네트 (14) C-IPP 혼입 계산 </C-IPP 농도를 측정하고, 시간의 함수로서 결과를 플로팅한다. 시간에 따른 순 14 C-IPP 통합의 증가가 예상된다. </li></ol>

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저자는 공개 할 것이 없습니다.

E. coli 세포에서 기능성 인간 DHDDS를 정제하기 위해 여기에 설명 된 프로토콜은 간단하고 효율적이어서 적절한 구조가 사용 가능 해지면 3-4 일 내에 단백질을 과발현하고 정제 할 수 있습니다. 단백질 정제를위한 그러한 프로토콜은 많은 질병 18 의 유전학에 관한 정보의 과다한 정보를 제공하는 게놈 시퀀싱의 돌파구를 고려할 때 특별한 의미가 있습니다. 따라서 단백질 ?...

Prenyltransferases 다중 축합 반응 1을 통해 아이소 펜 테닐 다이 포스페이트 (IPP)를 사용하여 알릴 포스페이트, 2 쇄 신장을 촉매하는 효소 군이다. E 형 효소 트랜스 이중 결합의 형성을 촉진하는 반면 3 Z 형 효소, 축합 반응, 시스 이중 결합의 형성을 촉매. -Prenyltransferases 시스 (시스 -PT, Z 형 효소) 고전 단 체인 (C 15), 중쇄 (C 50-55)에 자신의 제품의 사슬 길이에 따라 분류되며, 장쇄 (C 70-120 ) 4 . DHDDS (dehydrodolichyl diphosphate synthase)는 이소 펜 테닐 디 포스페이트 (IPP) 1을 이용한 다중 응축을 통해 farnesyl diphosphate (FPP, allylic diphosphate)의 사슬 신장을 촉매하는 진핵 장쇄 cis -PT입니다, 5 , 6 . 이것은 dehydrodolichyl 포스페이트의 형성을 초래하는 dolichylpyrophosphate 전구체 N - 결합 글리코 실화 단백질 1에 관련된 글리코 캐리어 분자로서의 C 55-100 polyprenyl 인산. Ashkenazi 유태인 중, DHDDS에있는 missense 비 보존 적 돌연변이 (K42E)는 염색체 열성 색소 성 망막염을 초래합니다 7 , 8 . 따라서, 본 프로토콜은 역학적 연구에 적합한 정제 된 DHDDS를 얻기 위해 개발되었다. Escherichia coli 는 재조합 단백질 발현을위한 가장 편리하고 비용 효율적인 숙주로 여겨지며, 따라서 가장 빈번하게 사용되는 숙주이기도합니다. 그러나 대장균 에서 이종성으로 단백질을 과발현 시키려 할 때, 단백질 특유의 고려가 이루어져야한다. 적절하게 접힌 상태에서 얻음대장균 으로부터의 재조합 단백질은 상이한 단백질의 독특한 특성으로 인해 단순한 문제가 아니다. 이러한 장애물을 극복하기 위해 수많은 접근법이 개발되었습니다. 여기에서, 단백질 융합, 최적화 된 배양 조건 및 코돈 - 최적화의 사용은 대장균 에서 기능적으로 활성 인 DHDDS의 과발현 및 정제를 허용하기 위해 사용되었다. 참고로, 단백질 융합하지 않고 효모 시스의 -PT를 과발현하는 이전 시도조차 세제 (12)의 존재에 불용성을 완료 할 예정 실패했습니다. 설명 된 프로토콜은 간단하고 비용 효과적이며 시간을 절약 할 수 있으며 기계 론적 연구에 적합한 DHDDS 준비를 얻을 수 있습니다. 서로 다른 종에서 cis -PT의 상 동성을 고려할 때,이 프로토콜은 다른 진핵 cis- PT에도 적용될 수 있다고 제안한다.

<table width="751">
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		<tr height="21">
			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

Prenyltransferases (PT)는 다중 응축 반응을 통한 isopentenyl diphosphate (IPP) 를 사용하여 알릴이 인산의 사슬 연신을 촉매하는 효소 군입니다. DHDDS (dehydrodolichyl diphosphate synthase)는 ispenetenyl diphosphate (IPP)와의 다중 축합을 통한 farnesyl diphosphate (FPP, allylic diphosphate)의 사슬 연신을 촉매하는 진핵 장쇄 cis -PT (축합 반응으로부터 cis 이중 결합을 형성 함)이다. DHDDS는 효소의 비 보존 적 돌연변이 (K42E)가 망막염 색소 침착 으로 이어 지므로 궁극적으로 실명으로 이끄는 생물 의학적으로 중요합니다. 따라서, 본 프로토콜은 역학적 연구에 적합한 대량의 정제 된 DHDDS를 얻기 위해 개발되었다. 여기에서, 단백질 융합, 최적화 된 배양 조건 및 코돈 최적화의 사용은 기능적으로 활성 인 DHDDS의 과발현 및 정제를 허용하기 위해 사용되었다. <em&gt; E이다. 콜리. 설명 된 프로토콜은 간단하고 비용 효율적이며 시간을 절약합니다. 상이한 종 사이의 cis -PT의 상 동성은이 프로토콜이 천연 고무 합성에 관여하는 것과 같은 다른 진핵 cis- PT에도 적용될 수 있음을 시사한다.

여기에 사용 된 구조 및 정제 과정에 대한 일반적인 개요가 그림 1에 나와 있습니다. 각 정제 단계에서 얻은 샘플을 그림 2 에 나타냈다 . 이 SDS-PAGE 분석은 DHDDS의 단계적 정제를 보여줌으로써 매우 정제 된 제품을 생성합니다. 도 3 은 정제 된 효소의 SEC 분석 결과로서, 단백질이 동종...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

에스 케리 치아 (Escherichia )로부터의 비 변성 조건 하에서 코돈 최적화 된 인간 시스 - 프레 닐 전이 효소의 과발현 및 정제를위한 간단한 프로토콜 콜라이 (coli )는 효소 활성 분석과 함께 기술된다. 이 프로토콜은 기계적 연구에 적합한 양 및 품질의 다른 시스 플 닐 트랜스퍼 라제 단백질의 생산을 위해 일반화 될 수 있습니다.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

overexpression-purification-human-cis-prenyltransferase-escherichia

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JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

6.5K 조회수.  Tel Aviv University.  에스 케리 치아 (Escherichia )로부터의 비 변성 조건 하에서 코돈 최적화 된 인간 시스 - 프레 닐 전이 효소의 과발현 및 정제를위한 간단한 프로토콜 콜라이 (coli )는 효소 활성 분석과 함께 기술된다. 이 프로토콜은 기계적 연구에 적합한 양 및 품질의 다른 시스 플 닐 트랜스퍼 라제 단백질의 생산을 위해 일반화 될 수 ?...

비디오: Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

연구

면역학 및 감염병학

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

특정 유전자의 Overexpression 또는 최저 후 라모스의 B 세포에서 체세포 Hypermutation을 평가

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

기본 인간 T 세포 piggyBac Transposon 시스템 변경

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic의 비 침습 모델<em&gt; 대장균</em&gt; 신생아 쥐에 감염

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

한 단계의 부정적인 크로마토 그래피 정제<em&gt; 헬리코박터 파이로리</em&gt; 호중구 - 활성화 단백질 과발현<em&gt; 대장균</em&gt; 배치 모드에서

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

밀도 Transposon 삽입 라이브러리를 사용 하 여 세균성 활용 Enterobacterial 종자와 같은 대장균 또는 Shigella flexneri 의 창조

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Site-Specifically 생성 하는 손쉬운 프로토콜 Acetylated 대장균 에서 단백질

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...