著者らは、ルイス・F・デルガディージョ氏(オハイオ大学化学・生体分子工学科)、マシュー・H・ウィリアムズ氏(オハイオ大学化学・生体分子工学科)、フィリベルト・セデノ・ローラン博士(ブリガム・アンド・ウィメンズ病院とハーバード大学医学部)に専門的な技術支援を求めています。著者らはまた、2011年冬のCHE 404/BME 504および秋2012 CHE 4830 /BME 5830学生(オハイオ大学化学・生体分子工学・生物医学工学専攻)に技術的な助言と議論を感謝したいと考えています。この研究は、国立科学財団(NSF)主要な研究計装助成金CBET-1039869(MMB)、NSF CBET-1106118(MMB)、皮膚財団研究助成金A050422(SRB)、国立衛生研究所(NIH)NCI助成金1R15CA161によって支援されました 830-01 (MMB)、NIHキルシュシュタイン-NRSAポスドク・フェローシップF32CA144219-01A1(SRB)、NIH/NCI R01CA173610(CJD)、NIH NCCAM助成金R01AT004628(CJD)。

1. プロテイン A のアフィニティを確認する
<ol><li>Fc発現タンパク質(組換え融合タンパク質またはIgG抗体、ここでは「タンパク質」と呼ばれる)が、タンパク質A膜吸着体を使用する前にタンパク質A14-17 に対して許容可能な親和性を有することを確認し、ストック溶液中にタンパク質の存在を試験する(例えば 、細胞培養上清を上清して1,000xgmin細胞に懸濁させた)。フローサイトメトリー、ウェスタンブロッティング、ELISA などを使用します。ラボの好みに基づいて、Fcのタンパク質機能および/または存在を検出します。理想的には、細胞培養上清中のタンパク質を定量化する(ステップ6)タンパク質A膜吸着量を超えないようにする。タンパク質が検出された場合に進みます。</li>
</ol>2. タンパク質A膜吸着剤および細胞培養上清を調製する
<ol><li>膜吸着を準備するためのメーカーの指示に従ってください。 空気を膜吸着器に入れないでください。膜吸着剤を介して浸透するすべてのソリューションは、室温で、0.22 μmフィルタを使用して事前にフィルタ処理する必要があります。 10 ml シリンジに 0.22 μmろ過ダルベックコのリン酸緩衝生理食塩水(DPBS)またはバッファーを充填し、エアバブルを排出します。DPBSを浸透して貯蔵溶液を除去し、使用直前に膜吸着を平衡化する。</li>
	<li>膜吸着体を通して灌流の直前に細胞培養上清をろ過し、真空駆動の0.22μm滅菌濾過ユニットを使用する。</li>
</ol>3. タンパク質A膜吸着体をロードする
<ol><li>吸気細胞培養は、ルアーロックシリンジ(30ml以上)に上清し、気泡を排出します。</li>
	<li>図 1に示すように、材料と機器を組み立てます。注射器を膜吸着器の入口に接続します。膜吸着口にフレキシブルチューブを取り付けます。必要に応じて、0.22 μmのシリンジフィルターをシリンジと膜吸着器の間に置きます。フラスコまたはボトルを使用して、吸着器から流れ(濾液とも呼ばれる)をキャッチします。</li>
	<li>シリンジポンプを所望の体積流量に設定しますが、メーカーの推奨流量(例えば 10ml/分)を超えないようにしてください。膜吸着剤を通して細胞培養上清を浸透させ、ビーカーまたは他の容器内を通って流れを集める。必要に応じて上澄みのあるシリンジをリロードします。</li>
	<li>必要に応じて、フローサイトメトリー、ウェスタンブロッティング、ELISAなどを使用してタンパク質の存在のために流れをテスト します。ラボの好みに基づいて。通常、フローを再変換する必要はありません。</li>
</ol>4. 膜吸着体からの溶出タンパク質
<ol><li>膜吸着体を10mlDPBSで洗浄し、非結合タンパク質を除去します。</li>
	<li>10~15mlの溶出緩衝液(例えばアミン系溶出緩衝液(pH2.8))を用いて所望の流量(例えば1ml/min)で膜吸着体から溶出タンパク質。中和バッファー(例えば1 Mトリス(pH 9.4))を含むチューブ内の溶出液を溶出量の10%(1.0〜1.5ml)で捕捉する。</li>
	<li>あるいは、1ml単位で溶出したタンパク質を100μlの中和バッファーを含むチューブに、10〜15ml溶出バッファーの総体積を使用した。好ましい特性評価方法を使用して、各分画がタンパク質の存在をテストします。</li>
	<li>メーカーの指示に従って膜吸着を再生します。パーフューズ 10 ml の 0.22 μm フィルター DPBS またはバッファーを選択し、1 N NaCl で 0.22 μm フィルター 50 mM NaOH の 10 ml、最後に 10 ml の 0.22 μm フィルター DPBS を使用します。膜吸着剤にDPBSで20%エタノールを充填し、4ºCで長期保存します。</li>
</ol>5. タンパク質の濃縮と透析
<ol><li>全ての溶出物(またはステップ4.3で決定したタンパク質を含む溶出画分)を10kDa分子量カットオフ遠心フィルターユニットに付着する。遠心分離の製造元の指示に従ってください。</li>
	<li>メーカーの指示に従って、選択したバッファーに対して少量透析ユニット(10 kDa分子量カットオフ)でリテン酸を透析します。タンパク質沈殿が懸念される場合、バッファーを透析材料に添加する必要があります。必要であれば、透析材料は、ステップ6まで冷蔵することができるが、防腐剤を使用しない長期保存はお勧めできません。</li>
</ol>6. 迅速なウェスタンブロッティング手順における精製物の定量化と特性化
<ol><li>SDS-PAGEにより、望ましい18-20の還元または非還元条件下で、選択したゲルに精製されたタンパク質およびFc基準を解決する。実行時間を最小限に抑えるには、ミディゲルではなくミニゲルを使用します。<ol>
<li>まだ知られていない場合は、帯信号がロードされる量(例えば 0.1-1mg)に関して線形に変化するFc規格範囲を決定する。精製タンパク質を定量化し、特性を示すために使用される同じゲル、走行条件、転移条件、試薬を使用します。</li>
			<li>DPBSで希釈したサンプルを含む精製タンパク質の複数のサンプル量をロードし、精製されたタンパク質バンド信号が画像分析におけるFc標準の線形シグナル範囲内にあることを確認する。</li>
		</ol>
</li>
	<li>半乾燥ブロッター13を用いて、不連続な緩衝系でゲルからポリビニリデン(PVDF)膜にタンパク質を移す。転送時間は通常5〜10分です。所望であれば、クーマシーは転写後にゲルを染色し、転写効率21を検証する。</li>
	<li>アルカリ性ホスファターゼ(AP)またはホースラディッシュペルオキシダーゼ(HRP)にコンジュゲートされた抗Fcまたは抗IgGを用いて免疫ブロットを行い、真空支援タンパク質検出システムを使用してラボの好みに基づいて実行します。免疫ブロット時間は、典型的には1時間未満である。</li>
	<li>選択した基質および方法(例えば AP基質および強化された化学発光)を用いて免疫ブロットを開発する。</li>
	<li>画像解析により精製タンパク質を定量化します。Fc規格からのバンド信号に対して線形回帰を実行します。R2>0.90の場合、ラインの方程式を使用してバンド信号からタンパク質の量を計算します。必要に応じてサンプル希釈を考慮します。タンパク質の定量はFcに基づいて行われるので、タンパク質とFcとの分子量差の値 <を調整します。</li>
	<li>機能的アッセイまたはフローサイトメトリー、ウェスタンブロッティング、ELISA などを介してFcを検出する方法を使用してタンパク質を検証する。ラボの好みに基づいて。</li>
	<li>タンパク質を所望の濃度に希釈したり、長期保存、凍結、またはそれ以外の方法でアリクォートする前に、精製されたタンパク質溶液に所望の防腐剤または安定剤を加えたりします。</li>
</ol>

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開示するものは何もありません。

本明細書に記載されたプロトコルは、製品の回収や膨張コストを著しく損なうことなく、ヒトFc(Gal-1hFc)を発現するキメラ融合タンパク質を迅速に単離し、特徴付けるために開発された。合理化されたワークフローの主要なコンポーネントは、分離のためのタンパク質A膜吸着体、およびウェスタンブロッティングによる特性評価のためのセミドリー転送および真空支援免疫ブロットです。
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免疫グロブリンG(IgG)Fc断片を発現する抗体および組換えタンパク質の単離は、細胞培養上清および希薄溶解細胞溶液から、プロテインAアフィニティークロマトグラフィーを使用して達成することができる。基礎科学・工学研究所および産業では、高い財政コストと長い処理時間1〜3にもかかわらず、多孔質タンパク質Aコーティングアガロース、ガラス、またはポリマービーズを詰めたカラムが最も一般的にアフィニティークロマトグラフィーに使用されています。アフィニティクロマトグラフィーは、ラボと産業の両方の設定で最大の費用と処理のボトルネックを表すことを高く評価されています2,4.その結果、プロセストレイン1,2,5-7からの製品全体の回復を犠牲にすることなく、コストと処理時間を短縮するために数多くの改良が開発されています。特に抗体および他のFc発現タンパク質の単離に対して有望なのは、A膜吸着体2,5,7-10のタンパク質Aを用いたアフィニティークロマトグラフィーである。カラムクロマトグラフィーにおけるFc捕捉は拡散に制限される(すなわち、Fc発現タンパク質は、内部細孔を介して拡散して、大部分のタンパク質Aに到達しなければならない)、カラムを横切る高圧降下を伴い、膜吸着体における大量輸送はバルク対流によって駆動され、拡散制限の回避をもたらす8,9,11,12。また、膜吸着量全体の圧力降下が低く、ビードパックカラム8,9,11,12に比べてより速い灌流流速度が可能となる。したがって、実験室規模の分離のために、膜クロマトグラフィーは、数時間の分離時間を短縮し、カラムクロマトグラフィーと比較して製品のキャプチャを増加させると予測されます。さらに、膜吸着物中のIgG吸着の数学的モデルは8,9,11,12を提案されており、エンドユーザーがラボでの性能を予測できるようにしています。
基礎科学・工学研究室のもう一つのボトルネックは、Fc発現タンパク質の特性化である。しかし、このようなウェスタンブロットを、特性評価アッセイを完了するための適切な方法の選択は、製品のテストに費やす時間を大幅に削減することができます。例えば、不連続緩衝システムにおけるセミドライ移動は、連続緩衝システム13におけるタンク内(湿式)移動の1〜2時間とは対照的に、SDS-PAGEゲルからポリビニルジフルオリジン(PVDF)膜へのタンパク質の電気泳動を数分で達成することができる。
ヒトIgGのFc断片を発現するキメラ融合タンパク質を単離し、特徴付ける迅速、安価、および有効なプロトコルが、本明細書に記載されている。ほとんどの装置は標準的な基礎生物医学、生物学、化学、および(生物)化学工学の実験室ですぐに利用でき、すべての試薬は市販されている。代表的な結果は、マウスガレクチン-1/ヒトFcキメラ融合タンパク質(Gal-1hFc)の単離および特性解析から生成されたが、合理化されたプロトコルは、抗体、Fcフラグメント自体、または他のFc融合タンパク質などのFcフラグメントを有する他の分子の単離に適用することができる。当社の改善は、標準的な方法と比較して、類似または改善された製品の回復を達成しながら、処理時間を劇的に短縮しました(4時間ほどですが、通常は20時間以上の作業時間と比較して〜9時間)。.

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

細胞培養上清および溶解細胞溶液からの生体分子の工業的規模への実験室規模の精製は、アフィニティークロマトグラフィーを使用して達成することができる。多孔質タンパク質Aアガロースビーズを使用したアフィニティークロマトグラフィーは、IgGのFc断片を発現する抗体および組換えタンパク質の実験室規模分離の最も一般的な方法であるが、時間とコストのかかるプロセスであり得る。時間と財務上の制約は、希薄溶液から数百マイクログラムからミリグラムのタンパク質を回収しなければならない小さな基礎科学ラボでは特に困難ですが、バイオ分離の専門知識を持つ高圧液体送達システムや人員へのアクセスが不足しています。さらに、製品の定量化と特性評価は、複数の稼働日にわたって処理時間を過度に長くし、費用(消耗品、賃金 など)を膨らませる可能性があります。したがって、Fcフラグメントを保有する抗体および他のタンパク質の検査尺度の分離および特性評価には、迅速で安価でありながら効果的なプロトコルが必要です。そのために、20時間以上の作業時間を必要とする従来の方法とは対照的に、経験の限られた技術スタッフが9時間未満(約1作業日)、4時間で完了できるプロトコルを考案しました。ほとんどの必要な装置は標準的な生物医学、生物化学、および(生物)化学工学の実験室ですぐに利用でき、すべての試薬は市販されている。このプロトコルを実証するために、細胞培養上清からヒトFc(Gal-1hFc)に融合したキメラマウスガレクチン-1がタンパク質A膜吸着体を用いて単離した代表的な結果が提示される。精製Gal-1hFcを迅速なウェスタンブロッティング分析手順を用いて定量し、フローサイトメトリーを用いて特徴付けた。合理化されたワークフローは、抗体などの他のFc発現タンパク質に対して変更したり、代替定量法や特性評価方法を組み込むために変更することができます。

Fc発現タンパク質の単離および特性評価のための合理化されたプロトコルは、希薄細胞培養上清からヒトFc(Gal-1hFc)に融合したキメラマウスガレクチン-1を処理するために日常的に使用されています。 図 2 のフローチャートは、プロトコルの各ステップのワークフローと時間を示しています。300mlの上澄み物の典型的なバッチの場合、溶出画分の任意のフローサイトメトリー試験が?...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

プロテインAアガロースビーズパックカラムを用いた従来のアフィニティクロマトグラフィーと比較して、プロテインA膜吸着体は、抗体およびその他のFc断片発現タンパク質の実験室規模の単離を大幅に高速化することができます。適切な分析と定量化方法により、タンパク質処理がさらに加速し、20時間以上の作業時間ではなく、1営業日で分離/特性解析を完了できます。

タンパク質A膜吸着体と合理化されたワークフローを用いたキメラヒトFc発現タンパク質の単離と特性評価

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Research

JoVE Journal

Bioengineering

7.1K ビュー.  Ohio University. プロテインAアガロースビーズパックカラムを用いた従来のアフィニティクロマトグラフィーと比較して、プロテインA膜吸着体は、抗体およびその他のFc断片発現タンパク質の実験室規模の単離を大幅に高速化することができます。適切な分析と定量化方法により、タンパク質処理がさらに加速し、20時間以上の作業時間ではなく、1営業日で分離/特性解析...

ビデオ: Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

生物工学

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

光音響流量測定法を用いて循環メラノーマ細胞の検出と分離

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：膜タンパク質のタンパク質 - タンパク質相互作用を識別するために、生化学的ツール<em&gt;インビボ</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

インテイン媒介人工タンパク質ヒドロゲルの合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

ケーススタディとして、タバコにおける一過性タンパク質発現：実験アプローチのデザインを使用した複雑系のキャラクタリゼーション

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

神経組織再生のための可溶性および不溶性ビオチン化タンパク質の発現、分離、および精製

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

発光共鳴エネルギーは、哺乳動物細胞で発現膜タンパク質の立体構造変化を研究するために転送

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

インデントと引張試験を用いたヒト軟部組織の生体力学的特性評価

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

マクロを含む光の組織ファントムの作製と評価

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...