この研究はイスラエル科学財団の構造細胞生物学（I-CORE）のためのセンター（1775/12）およびイスラエル科学財団のグラント1721/16および2338/16（YH）および825/14（DK ）。 DKへのFields Estate Foundationのサポートは高く評価されています。この研究は、テルアビブ大学サックラー医学部のMD卒業要件の部分的な履行においてIlan EdriとMichal Goldenbergが行った。

1。 大腸菌におけるcis- PTの過剰発現のクローニング<ol><li> 109aaチオレドキシン（TRX）タンパク質17と融合したタンパク質配列および完全長cis -PTの大腸菌コドン最適化18コード配列のクローニングおよび高レベル発現のために設計されたpET-32b発現ベクターを得る。 </li><li> シスの上流にTEV-プロテアーゼ（タバコエッチウイルスプロテアーゼ）切断部位（ENLYFQ / G、「/」は切断点を示す） 9、続いて短い柔軟性リンカー（切断部位接近性を高めるためのSGSGSG） -PT配列（ 図 1A ）。また、クローニングに適した5 &#39;および3&#39;制限部位を加える。 </li><li>標準的なライゲーション技術を用いて、合成されたDNA構築物をpET-32bベクターにクローニングする10 。 </li></ol> 2。過大評価大腸菌におけるヒトDHDDSのssion <ol><li>形質転換大腸菌 T7は、TRX融合DHDDS構築物を有するコンピテント細胞を、製造者の指示に従って発現する。 </li><li>形質転換細胞をアンピシリン選択条件（200mg / L）下のLB寒天上の製造者の指示に従ってプレートする。 </li><li> 200mg / Lのアンピシリンを含む250mLフラスコスターター培養物中の100mLのLB培地に選択されたコロニーを接種し、200rpmで一定に振盪しながら37℃で一晩インキュベートする。 </li><li> 100mg / mLの濃度の2×YT培地（16g / Lトリプトン、10g / L酵母エキス、5g / L NaCl）1.5Lを含む2つの5Lフラスコ（フラスコ当たり40mL）に、80mLの培養物を接種する。 Lアンピシリン。 </li><li> OD 600 nm = 0.5に達するまで180 rpmで一定に振とうしながら37℃で培養物をインキュベートする。 </li><li>温度を16℃に下げ、0.5mMイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を添加して細胞を誘導する。コン同じ条件下でタンパク質発現のために16〜20時間インキュベートする。 </li><li>遠心分離（10,000× g 、10分間）により細胞を回収する。 注意：ここではプロトコルを一時停止することができます。細胞ペレットは精製するまで-80℃に保つ必要があります。 </li></ol> 3。ヒトDHDDSの精製<ol><li>バッファーA（25mM Tris-HCl、pH 7.5,150mM NaCl、0.1％Triton X-100,10mMβ-メルカプトエタノール）、1μg/ mL DNase I、およびプロテアーゼ阻害剤混合物に細胞を再懸濁する。 </li><li>ガラステフロンホモジナイザーを用いて細胞をホモジナイズする。 </li><li> 15,000 PSI 11 - 12000でマイクロフルイダイザーまたは同等のものを使用して細胞を破壊。 </li><li> 4℃で45分間40,000 xgで細胞ライセートを遠心分離する。可溶性画分を含む上清を回収する。 </li><li>上清を5~10mLのコバルト固定化金属アフィニティークロマトグラフィー（Co 2+ -IMAC） 12カラムに10続いて緩衝液Aおよび10mMイミダゾールで十分に洗浄して、非特異的タンパク質結合を減少させた。 </li><li> 250mMイミダゾールを補充した緩衝液Aで過剰発現したタンパク質を溶出する。 </li><li>バッファーAで平衡化した53mL分取脱塩カラムを用いてイミダゾールを除去する。 </li><li>溶出されたタンパク質に6xHis標識TEVプロテアーゼ（タンパク質50mgあたり1mgのTEVプロテアーゼ）を添加して、6xHis標識TRX融合タンパク質を4℃で一晩除去する。 </li><li>開裂したタンパク質を、5mMイミダゾールを補充した緩衝液Aで平衡化したCo 2+ -IMACカラムにロードし、切断された6xHis標識TRX融合タンパク質およびTEVプロテアーゼを除去する。 </li><li> 30kDa分子量カットオフ遠心フィルターを使用して、フロースルーを集め、3〜4mLに濃縮する。 </li><li>最終精製のために緩衝液Aで平衡化されたsuperdex-200サイズ排除クロマトグラフィーカラムに濃縮タンパク質をロードする。タンパク質は二量体（約76kDa）として溶出する。 </&gt; <li>溶出プロファイルとカラム較正曲線を比較することにより、関連する画分を選択する。 </li><li> SDS-PAGEを用いて調製物の純度を評価する。 </li><li>下流の実験に必要なタンパク質濃度を決定し、液体窒素中の精製され濃縮されたタンパク質のフラッシュ凍結アリコートを使用し、使用するまで-80℃で保存する。 </li></ol> 4。分析サイズ排除クロマトグラフィー（SEC） <ol><li>凍結融解サイクルに耐えるタンパク質の能力を確実にするために、凍結タンパク質のアリコートを解凍し、21,000× gで10分間遠心分離して不溶性凝集物を除去する。上清を集める。 </li><li>超高性能液体クロマトグラフィーシステム13を用いて、25mM Tris-HCl、pH7.5,150mM NaCl、0.02％Triton X-100,10mMβ-メルカプトエタノールで予め平衡化したsuperdex-200分析カラムにタンパク質をロードする。 14 。 </li><li>トリプトファンフルオルをモニターするscenceタンパク質溶出溶出プロファイルを検出するために、（λのEX = 280nmで、λEMは、350nmでの=）。分子量評価のための有効な検量線があることを確認してください。このような曲線はSECカラム製造業者から入手できます。 </li></ol> 5。酵素動力学-時間依存性活性15、16、17 <ol><li>精製されたタンパク質の活性を確実にするために、5μMの精製DHDDSを10μMのFPPおよび50μMの14 C-IPPと混合して、緩衝液A中で0.5mMのMgCl 2を用いて22〜30℃で反応を開始させる。 注：正確な反応条件は、DHDDSとは異なるシス -PTの調製によって変化し得る。さらに、DHDDSの活性はまた、温度および[Mg 2+ ]に依存する。 注意：14 C-IPPが放射性で、地域内の放射線安全規則に従って使用されるべきです。 </&gt; <li> 0,2,4および6時間後に15μLのサンプルを0,2,4および6時間後に取り出し、緩衝液Aを補充した20mM EDTA（最終濃度10mM EDTA）を添加することにより反応を即座に停止させた。 </li><li> H 2 O飽和1-ブタノール1mLを添加し、完全にボルテックスして反応生成物を抽出する。 注：プロトコルはここで停止することができ、サンプルは後で読むために保管することができます。 </li><li>サンプルにシンチレーションカクテルを加え、シンチレーションカウンターを用いて、 14 Cを含むブタノール相の反応生成物を、反応混合物からの放射能（全放射能を表す）と共に定量する。 </li><li>各時点からの0時間サンプルを用いて測定されたバックグラウンド測定値を差し引き、使用された14 C-IPPのパーセントを計算する。 </li><li>合計時点14から利用率を計算することにより、各時点での正味14 C-IPP取り込みを計算する。 </その結果を時間の関数としてプロットした。 14 C-IPPの正味の組み込みの増加が予想される。 </li></ol>

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著者は何も開示することはない。

大腸菌細胞内で機能的ヒトDHDDSを精製するために本明細書に記載されたプロトコールは、簡便かつ効率的であり、適切な構築物が入手できれば3〜4日でタンパク質を過剰発現および精製することができる。タンパク質精製のためのそのようなプロトコールは、多くの疾患18の遺伝学に関する情報の過多を提供するゲノムシーケンシングのブレークスルーを考慮すると特...

プレニルトランスフェラーゼは、複数の縮合反応1 を介して、イソペンテニル二リン酸（IPP）を使用して、アリル二リン酸、2の鎖の伸長を触媒する酵素の一群です。 Z型酵素は、縮合反応からシス二重結合の形成を触媒するが、E型酵素はトランス二重結合形成を触媒する3 。 cis-プレニルトランスフェラーゼ（ シス -PT、Z型酵素）は、その鎖長に応じて、短鎖（C 15 ）、中鎖（C 50-55 ）、および長鎖（C 70-120 ） 4 。 DHDDS（dehydrodolichyl二リン酸シンターゼ）は、イソペンテニル二リン酸（IPP）1と、複数の縮合を介して、ファルネシル二リン酸（FPP、アリル二リン酸）の鎖伸長を触媒する真核生物の長鎖シス -PTあります、5、6。これはdehydrodolichylジホスフェート、dolichylpyrophosphateの前駆体、N結合型タンパク質のグリコシル化に関与する1グリコシル担体分子としてのC 55から100ポリプレニル二リン酸の形成をもたらします。アシュケナージ系ユダヤ人、常染色体劣性網膜色素変性症7,8におけるDHDDS結果におけるミスセンス非保存的変異（K42E）のうち。従って、本プロトコールは、機械的研究に適した精製DHDDSを得るために開発されたものである。 エシェリヒア・コリは、組換えタンパク質発現のための最も便利でコスト効率の良い宿主であると考えられ、従って、最も頻繁に使用される宿主でもある。しかし、 大腸菌でタンパク質を異種的に過剰発現しようとする場合、タンパク質特異的な検討が必要である。正しく折りたたまれた状態で取得する大腸菌由来の組換えタンパク質は、異なるタンパク質の異なる特性のために単純な問題ではない。これらのハードルを克服するための多くのアプローチが開発されている。ここでは、 大腸菌で機能的に活性なヒトDHDDSの過剰発現および精製を可能にするために、タンパク質融合、最適化培養条件およびコドン最適化の使用を使用した。注目すべきは、タンパク質融合なしで酵母cis -PTを過剰発現する以前の試みは、界面活性剤の存在下でさえ完全な不溶性のために成功しなかった12 。記載されたプロトコールは、単純で費用効果が高く、時間を節約し、機械学的研究に適したDHDDS調製物を得ることを可能にする。異なる種間のシス -PTの相同性を考慮すると、このプロトコールは他の真核生物シス -PTにも適用できることを示唆している。

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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>
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		<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>
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		<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

プレニルトランスフェラーゼ（PT）は、複数の縮合反応によるイソペンテニル二リン酸（IPP） を用いたアリル二リン酸の​​鎖伸長を触媒する酵素群である。 DHDDS（デヒドロドキリチルジホスフェートシンターゼ）は、イソペンテニル二リン酸（IPP）との多重縮合を介してファルネシル二リン酸（FPP、アリル二リン酸）の鎖伸長を触媒する真核生物の長鎖シス -PT（縮合反応からのシス二重結合を形成する）である。 DHDDSは生物医学的に重要であり、酵素中の非保存的突然変異（K42E）が色素性網膜炎をもたらし、最終的には失明に至る。したがって、本プロトコールは、機構的研究に適した精製DHDDSを大量に取得するために開発されたものである。ここでは、機能的に活性なヒトDHDDSの過剰発現および精製を可能にするために、タンパク質融合、最適化培養条件およびコドン最適化の使用を使用した。 <em&gt; Eである。大腸菌。記載されたプロトコルは、単純で、費用効果が高く、時間を節約する。異なる種間のシス -PTの相同性は、このプロトコルが天然ゴム合成に関与するものなどの他の真核性シス -PTにも適用できることを示唆している。

ここで使用したコンストラクトの概要と精製プロセスを図1に示します 。各精製工程で得られた試​​料を図2に示す。このSDS-PAGE分析は、DHDDSの段階的精製を示し、高度に精製された生成物をもたらす。 図3は、精製酵素の分析SECの結果を示し、タンパク質がホモ二量体としての?...

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

Escherichiaからの非変性条件下でのコドン最適化されたヒトcis-プレニルトランスフェラーゼの過剰発現および精製のための簡単なプロトコール 大腸菌は 、酵素活性アッセイと一緒に、記載されています。このプロトコールは、機構的研究に適した量および質の他のシスプレニルトランスフェラーゼタンパク質の産生のために一般化することができる。

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</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

Research

JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

6.5K ビュー.  Tel Aviv University.  Escherichiaからの非変性条件下でのコドン最適化されたヒトcis-プレニルトランスフェラーゼの過剰発現および精製のための簡単なプロトコール 大腸菌は 、酵素活性アッセイと一緒に、記載されています。このプロトコールは、機構的研究に適した量および質の他のシスプレニルトランスフェラーゼタンパク質の産生...

ビデオ: 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.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

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

免疫・感染学

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.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

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.

特定の遺伝子の過剰発現やノックダウン後にラモスB細胞に体細胞高頻度変異の評価

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.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

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.

神経病原の非侵襲的モデル<em&gt;エシェリヒア·コリ</em&gt;新生ラットにおける感染

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.

ワンステップの負のクロマトグラフィー精製<em&gt;ヘリコバクター・ピロリ</emで過剰発現&gt;好中球活性化タンパク質<em&gt;エシェリヒア・コリ</em&gt;バッチモードでの

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.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

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.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

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.

密なトランスポゾン挿入ライブラリを使用して細菌活用腸内細菌系統など、大腸菌や赤痢菌の作成

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.

大腸菌Site-Specifically アセチル化蛋白質を生成する安易なプロトコル

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