This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

大規模な一過性トランスフェクションのためのプラスミドDNAのミリグラム量の1生産<ol><li>制限部位は、クローニング、またはその他の適切な技術を用いて、高コピー数の哺乳動物発現ベクターに目的のタンパク質をクローニングします。 <ol><li>最適な結果を得るために、組み込まれているC末端の6Hisタグ、強力なプロモーターKozak配列および最適化された分泌シグナルpHLsec 7ベクトルを、使用しています。 </li></ol></li><li>コンピテント細胞にプラスミドを変換します。 <ol><li>プラスミドDNA1μgのにコンピテント大腸菌細胞の20μlを添加し、氷上で30分間インキュベートします。 </li><li> 35秒間42℃で熱ショック細胞は、その後2分間氷上でインキュベートします。 </li><li>微生物の増殖培地（SOC）の300μlを添加して、220rpmで振とうしながら、45分間37℃でインキュベートします。 </li><li>適切な抗生物質選択と寒天プレート上で平板細胞。 <ol><li>プラスミドはpHLsecベクターである場合は、100μg/ mlのカルベニシリンを使用してください。 </li></ol></li></ol></li><li>ルリアブロス（LB）メディアの250ミリリットル中の培養コロニーは、220 rpmで振とうしながら、37℃で100μg/ mlの抗生物質（カルベニシリン）、O / Nで補充。 </li><li>製造業者のプロトコルに従ってハイスピー​​ドプラスミドマキシキットを使用して培養物からDNAを精製します。 <ol><li>代わりにバッファTEの、バッファEB（10mMのトリス-Cl、pH8.5）中のDNAを溶出します。 </li><li> -20℃でのトランスフェクションおよびストアに必要な量で精製されたプラスミドを等分。 </li></ol></li></ol> 293F細胞の2大規模培養および一過性トランスフェクション<ol><li>グルタミン10ml及び5 mlのペニシリン/ストレプトマイシン（両方とも100倍）を用いて追補1 L 293F媒体。 4℃で保存します。 5 mlのペニシリン/ストレプトマイシンを無血清条件で十分な強度であり、減少した抗生物質の濃度は、タンパク質収率を改善するトランスフェクション時に細胞生存率を改善します。 </li><li> 1Lのポリカーボネート中の300ミリリットル培地で培養293F細胞は、通気キャップで三角フラスコバッフル8％CO 2、37℃での、標準的な組織培養インキュベーター中で振盪しながら。 </li><li> 5×10 5 / mlの密度、トランスフェクションの前日に細胞を希釈します。 </li><li> 293F培地における細胞ブーストw / vの2％の10％の容量を追加することにより、トランスフェクション、補充培地の日。 <ol><li> 500ミリグラム/ dLのグルコース濃度を達成するために必要に応じて、製造者の指示及び使用サプリメントによるグルコースモニタを使用してグルコース濃度を測定します。 </li></ol></li><li>タンパク質のグリコシル化を制御するには、この段階でキフネンシン（1 / mlの最終濃度）を追加します。 </li><li> 1×10 6個の細胞あたり1μgのプラスミドに必要なDNAの量を計算します。無菌条件下で、5 mlの無血清培地中でDNAを希釈します。 </li><li>トランスフェクション試薬μlの2当たり1μgのプラスミドに必要なトランスフェクション試薬の量を計算します。無菌条件下で、5 mlの無血清培地中でトランスフェクション試薬（PEI-TMC-25）を希釈します。 </li><li>追加1ミリリットル単位でDNA溶液へのトランスフェクション試薬は、穏やかに混合します。形成するための試薬-DNA複合体を室温で30分間インキュベートします。その後、滴下方式で細胞上に溶液を加えます。 </li><li>トランスフェクションされた細胞は、72〜96時間タンパク質を発現することを許可します。 〜10％の容量セルブースト毎日メディア、または必要に応じてとの補足は、グルコースが400〜600ミリグラム/デシリットルを読み続けます。 </li></ol> 3.精製<ol><li>細胞をペレット化した後、上清を収集するために1300×gで20分間、遠心分離フラスコに培養物を遠心分離しデカントします。必要な場合は、2番目の時間をスピンおよび/または上清を明確にするために、0.22μmのフィルターを使用しています。 </li><li> 10％容積の10倍のNi-NTA結合バッファー追加（1.5 MのNaCl、0.5 K 2 HPO 4、0.1 MトリスpHは8.5、50mMのイミダゾール）。 </li><li>カラム内のNi-NTAスラリーを2mlを添加し、1×結合緩衝液の10カラム容量（CV）で平衡化することによって、重力カラムを準備します。可能な場合は、4℃の部屋にすべての列の手順を実行します。 Alternativエリー、チルタンパク質とカラム工程の前に、氷の上のすべてのバッファ、および蛋白質を維持し、収集したフロースルー氷上。 注：のNi-NTAスラリーは、50体積％の樹脂であり、製造者述べ結合能力は、50 mg / mlです。 Ni-NTAビーズを複数の用途のために再充電することができます</li><li>樹脂上で上清をフローとフロースルーを収集します。この手順を繰り返します。 </li><li>洗浄緩衝液（300mMのNaCl、50mMのK 2 HPO 4、20mMのイミダゾールをpH8）の10 CVで洗浄しました。 </li><li>溶出緩衝液（300mMのNaCl、50mMのK 2 HPO 4、250 mMイミダゾールのpHは8）の5 CVでタンパク質を溶出します。 </li><li>脱グリコシル化が必要な場合： <ol><li> 0.5ミリリットルの最終容量のために、遠心濃縮器を用いて、0.43ミリリットルに溶出液を濃縮します。沈殿物を形成する場合は、16,000×gで4℃で遠心分離することにより、任意の破片をペレット化。 </li><li> 500 mMのクエン酸ナトリウムのpHが5.5の50μLを加えます。 </li><li> EndoHf20μlの（1×10 6 U / ml）を追加します。室温で2時間インキュベートします。 ありませんE：酵素が濃縮されたタンパク質が凝集するおそれがある、37℃で最適に動作します。脱グリコシル化は、SDS-PAGEまたはイムノブロットによって評価すると、RTインキュベーションを拡張し、不完全です。酵素は4℃で活性を有していません。 </li><li>リン酸緩衝生理食塩水（PBS）または最終貯蔵緩衝液で洗浄アミロース樹​​脂3倍：EndoHfを削除します。 4°Cで1時間樹脂を有するタンパク質をインキュベートします。ビーズをペレット化し、上清を回収し千×gで5分間回転さ。 </li><li>適切な分子量カットオフ遠心フィルターを用いてタンパク質を濃縮し、保存緩衝液（150mMのNaCl及び20mMのHEPES pH7.5）中に緩衝液交換。 </li></ol></li></ol>

<ol>
	<li>Meyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-host+expression+system+for+recombinant+production+of+challenging+proteins.">Multi-host expression system for recombinant production of challenging proteins.</a> PLoS One. 8, e68674 (2013).</li><li>Chang, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Rich, J. R., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+the+production+of+homogeneous+human+glycoproteins.">Emerging methods for the production of homogeneous human glycoproteins.</a> Nat Chem Biol. 5, 206-215 (2009).</li><li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-2+promotes+macrophage+survival+and+lung+disease+after+respiratory+viral+infection.">TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.</a> J Exp Med. 212, 681-697 (2015).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitigated+cytotoxicity+and+tremendously+enhanced+gene+transfection+efficiency+of+PEI+through+facile+one-step+carbamate+modification.">Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.</a> Adv Healthc Mater. 2, 1304-1308 (2013).</li><li>Elbein, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosidase+inhibitors:+inhibitors+of+N-linked+oligosaccharide+processing.">Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.</a> FASEB J. 5, 3055-3063 (1991).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Kelker, M. S., Debler, E. W., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+mouse+triggering+receptor+expressed+on+myeloid+cells+1+(TREM-1)+at+1.76+A.">Crystal structure of mouse triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.76 A.</a> J Mol Biol. 344, 1175-1181 (2004).</li><li>Kober, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+crystallization,+and+preliminary+crystallographic+analysis+of+wild-type+and+mutant+human+TREM-2+ectodomains+linked+to+neurodegenerative+and+inflammatory+diseases.">Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.</a> Protein Expr Purif. 96, 32-38 (2014).</li><li>Yurtsever, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-cleavage+of+human+CLCA1+protein+by+a+novel+internal+metalloprotease+domain+controls+calcium-activated+chloride+channel+activation.">Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.</a> J Biol Chem. 287, 42138-42149 (2012).</li><li>Sala-Rabanal, M., Yurtsever, Z., Nichols, C. G., Brett, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+CLCA1+modulates+TMEM16A+to+activate+Ca-dependent+chloride+currents+in+human+cells.">Secreted CLCA1 modulates TMEM16A to activate Ca-dependent chloride currents in human cells.</a> Elife. 4, (2015).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2, 2212-2221 (2007).</li></ol>

The authors declare that they have no competing financial interests.

HEK 293F細胞は、翻訳後修飾を必要とするタンパク質の強固な生産を提供しています。このシステムは、それ以外の場合は、より日常的表現のツールを使用して存在しないことになるジスルフィド、グリコシル化、およびリン酸化を含むネイティブに折りたたまれたタンパク質の迅速かつスケーラブルな発現を可能にします。また、このシステムは、単に複数のプラスミドの同時トランスフェ?...

原子レベルでタンパク質の構造を理解することは、生物学的経路および疾患の分子基盤を解明する鍵となります。 X線タンパク質結晶学は、高分子構造を決定するための最も広く使用される/適用可能な方法です。この方法の主な課題は、適切に折り畳まれた、純粋なタンパク質の十分な量を得ることです。これは、特に、特定の翻訳後修飾を受ける哺乳類分泌タンパク質を扱う問題となります。 細菌発現タンパク質は、タンパク質データバンク1に堆積し結晶化するタンパク質の主要な源です。それらは安価、高速であり、典型的にはタンパク質の高収量を産生するための細菌発現系は、主に好ましいです。しかし、細菌中で発現哺乳動物タンパク質の細胞外ドメインは、多くの場合、適切にケースがリフォールディングし、大規模な精製工程をfoを均一に得るために必要とされる、折り畳まれていませんldedタンパク質。さらに、多くの哺乳動物タンパク質は、適切なフォールディング2を達成するために 、翻訳後グリコシル化を必要とします。酵母または昆虫細胞での発現およびグリコシル化は、フォールディングの問題を克服することができるが、グリコシル化を含む翻訳後修飾は、不正確なまたは不均一な修飾を有するタンパク質をもたらす、哺乳動物細胞3のものとは大きく異なります。 哺乳動物細胞は、適切な翻訳後修飾や折りたたみを確保するために必要なすべての分子機構を表現します。しかしながら、これらの発現系は、典型的には、限られた収率及び試薬および消耗品のコスト高のために、ほとんどの研究室で好ましくない。ポリエチレンイミン（PEI）は、標準的なトランスフェクション試薬は、比較的安価であるが、細胞培地、DNA、及び培養装置におけるコストの増加をもたらす、かなりの細胞毒性および低いトランスフェクション効率を課します。 PEIへの多くの選択肢が非常に高価です。我々は対処します改善された細胞培養ツールの組み合わせを記述し、化学的に単一工程精製し、分泌された哺乳動物タンパク質の発現のための迅速で比較的安価な方法に対するPEI修飾することによって、これらの問題。この強力な方法は、機能的および生化学的研究4のために十分な収率を与え、場合によっては、さらに精製せずに結晶化を受けやすいタンパク質をもたらします。 このプロトコルは、懸濁液中で増殖させたヒト胚腎臓（HEK）293F細胞における分泌哺乳動物タンパク質のための発現と歩留まりを最大化するために、いくつかの手法について説明します。トランスフェクション効率（及び費用）、タンパク質産生および精製は、すべて非常にこのプロトコルに従うことによって強化されます。シングルステップ開環反応（文献5に詳細に記載さPEI-TMC-25、合成および特性）を介してカルバメートの付加により修飾PEIが大きく、カチオン性の細胞毒性を減少させ、トランスフェクション効率を向上させます膜破壊し、それに応じて、実験のコストを削減します。さらに、細胞生存率およびタンパク質の発現が大幅にグルコースおよびビタミンを供給するための培養栄養補助食品の添加によって改善されます。重要なことには、グリコシル化タンパク質の産生、キフネンシンによる治療、マンノシダーゼIの非毒性の化学阻害剤のための代わりに単一のN-アセチルグルコサミンを有するタンパク質を生成するためにエンドグリコシダーゼEndoHfによって除去することができる定義された、未成熟グリカンを有するタンパク質を生成します完全長のN-結合グリカン6。最後に、無血清、化学的に定義された培地中へのタンパク質の分泌は、構造的および生化学的研究のための迅速かつ容易な精製を可能にします。シングルステップニッケル - ニトリロ三酢酸（Ni-NTA）樹脂精製上清中の種の汚染の大部分を除去し、ある場合には、結晶化のために十分な純度のタンパク質を得ることができます。

<table>
	<tbody>
		<tr>
			<td>Culture Flasks</td>
			<td>GeneMate</td>
			<td>F-5909B</td>
			<td></td>
		</tr>
		<tr>
			<td>293 Freestyle Media</td>
			<td>Gibco/Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco/Life Technologies</td>
			<td>35050-061</td>
			<td>Use in place of Glutamine</td>
		</tr>
		<tr>
			<td>Hype 5 transfection reagent</td>
			<td>Oz Biosciences</td>
			<td>HY01500</td>
			<td></td>
		</tr>
		<tr>
			<td>293fectin transfection reagent</td>
			<td>Life Technologies</td>
			<td>12347019</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI transfection reagent</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow&#160;</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo Hf</td>
			<td>NEB</td>
			<td>P0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose Resin</td>
			<td>NEB</td>
			<td>E8021S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Boost R05.2</td>
			<td>HyClone</td>
			<td>SH30584.02</td>
			<td>Cell Culture Supplement</td>
		</tr>
		<tr>
			<td>GlucCell</td>
			<td>CESCO Bioengineering</td>
			<td>DG2032</td>
			<td>Glucose Monitoring System</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>519850.91</td>
			<td>Serum Free Medium for DNA transfection</td>
		</tr>
		<tr>
			<td>Luria Broth (LB Media)</td>
			<td>Life Technologies</td>
			<td>10855-001</td>
			<td></td>
		</tr>
		<tr>
			<td>GC10 Competent Cells</td>
			<td>Sigma-Aldrich</td>
			<td>G2919</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

ここで骨髄細胞2（hTREM2、残基19から132）上に発現ヒトタンパ​​ク質誘発性受容体から分泌される13 kDaの免疫グロブリン（IG）ドメインに適用され、この発現系の結果に従います。 TREM2は2つのジスルフィド結合と2つのN結合型グリコシル化部位を有する単一の細胞外Igドメインを含むI型膜貫通タンパク質です。多くの他のIgドメインタンパク質8とは異なり、TREM2は、細菌封入体9</su...

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

効率的な哺乳動物細胞発現および結晶化のための細胞外糖タンパク質のシングルステップの精製

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

efficient-mammalian-cell-expression-single-step-purification

Research

JoVE Journal

Biology

13.7K ビュー.  Washington University School of Medicine. This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

ビデオ: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳類のリンカーヒストンサブタイプの発現解析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...