著者らは、この仕事をサポートして資金調達のためにアクロン大学を承認したいと思います。

著者らは、開示することは何もありません。

組換えタンパク質工学は多くの分野にまたがる非常に強力な技術である。これは、カスタム設計のタンパク質の高収量の産生を可能にする、費用対効果の高い、調整可能な比較的簡単な手順である。これは、設計および標的タンパク質を発現することは必ずしも容易ではないことに留意することが重要である。基底発現および組換えタンパク質の安定性を、ベクターの特定の選択に依存E.?...

タンパク質は、最終的に適切な組織形成、組織を導く、多くの生物学的機能に関与している生体分子の広い範囲をカバーする。これらの分子は、人体内の均衡を維持し、規制アップ及び/またはダウンレギュレーション遺伝子および他のタンパク質を制御するシグナル伝達経路の数千を開始する。単一のタンパク質の破壊は、壊滅的な障害または疾患の発症につながることができた信号のこのウェブ全体に影響します。ラボで個々のタンパク質を設計することは、これらの悪影響に対処するための一つの解決策を提供しており、低分子薬物に代わるものを提供しています。 1977年には、14個のアミノ酸ソマトスタチン配列をコードする遺伝子は、E.を使用して作成された第一の操作されたポリペプチドの一つであった大腸菌 1。まもなく1979年の後、インスリンは、形質転換、プラスミドpBR322にクローニングし、発現させ、精製した2。それ以来、組換えタンパク質は、解像度の複数のフィールドに自分の影響力を拡大しているearch などの生体材料、ドラッグデリバリー、組織工学、バイオ医薬品、農業、産業用酵素、バイオ燃料として（レビューは参考文献3-8を参照してください）。これは、技術には、目的のために、化学的部分またはタンパク質配列の特定のアプリケーションの添加を介して提供し、これらに限定されないが、タンパク質同定、安定化および精製を標的とすることが汎用性によるところが大きい。 組換えDNA技術によって、組換えタンパク質は、哺乳動物、植物、昆虫、酵母、真菌、または細菌を含む真核生物および原核生物宿主系の種々の発現させることができる。各ホストは異なる利点を提供し、通常は最高のシステムは、タンパク質機能、収率、安定性、全体的なコスト、および拡張性に基づいて決定される。細菌細胞は、多くの場合、真核生物宿主（ すなわちグリコシル化、ジスルフィドブリッジ、Eを提供する翻訳後修飾のメカニズムを欠いている<html> TC。）5。しかしながら、これらのホストは、典型的にはより高価で時間のかかる09であり、その結果、哺乳動物および昆虫システムは、通常、真核生物のタンパク質のより良好な相溶性および発現をもたらす。従って、E.細胞は、安価な増殖条件および遺伝子発現機構において急速に拡大しているため、大腸菌は、よく理解されている5,9は、我々の発現システムのための好まれる宿主である。さらに、このシステムは、翻訳後修飾10の欠如にもかかわらず、機能的なタンパク質の生産を目的とし、その結果のためのスケールアップが容易である。 E.この株は、高い形質転換効率に基づいた優れたプラスミド収量を提供していますので、 大腸菌 K12株は、クローニングのために、このプロトコルで選択される。さらに、E.この宿主株は、制御されたタンパク質の発現および安定性11を提供するT7 RNAポリメラーゼ遺伝子が含まれているため、 大腸菌 BL21株は、発現のために利用される。 10トン &quot;&gt;ホスト選択後、さらなる注意が組換えタンパク質を合成することは、バクテリオファージT7転写および翻訳シグナルの指示の下でクローン化され、標的DNA配列で始まる。選択され、制御されたタンパク質の発現を促進するために理想的な発現ベクターを選択する際に注意しなければならず発現は、T7 RNAポリメラーゼ遺伝子12の染色体コピーを含有する宿主細胞において誘導される。プラスミドベクターpBR322に由来するこれらのベクターは、（レビューのためには、13を参照する参照）、しっかりと最初にStudierら14によって開発されたT7プロモーターによって制御され、付加的に提供されているlacオペレーター 、およびlacリプレッサー（LAC1）15,16の包含を介して制御組換えタンパク質工学では、この発現系は、異なる標的DNA配列を挿入することにより、所望のタンパク質の特定のアミノ酸配列を調整するまたは融合タンパク質を作成する機能を提供合わせたドメインから構成されるシングルタンパク質からのS。さらに、いくつかのベクターシリーズはN又はC末端上に配置するペプチドタグ修飾を含む。我々の設計のために、ヒスチジン（His）タグは、精製の ​​ためのDNA標的配列に加え、15個のアミノ酸配列は、ビオチン化biotinylatable 17,18のために含めた。このプロトコルでは、アンピシリン耐性遺伝子を含むプラスミドは、我々のbiotinylatable融合タンパク質配列を運ぶために選択した。発現はT7のlacプロモーターを介してこのベクター中に制御され、容易にイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）で誘導される。 テスト式（小スケールの培養物）を、精製手順のいずれかを処方するために可溶性または不溶性の形態で発現することができる標的タンパク質の存在および溶解度を決定するために使用される。細菌細胞内で発現される可溶性タンパク質は、その天然の構造体19を維持するために自発的なフォールディングを受けることになる。通常、ネイティブ構造は熱力学的に有利である。多くの場合、宿主の代謝活性は、不溶性タンパク質産生および不溶性タンパク質凝集体からなる封入体の形成をもたらすシステムにストレスを与え、標的タンパク質に資するものである。従って、標的タンパク質は20生物学的に不活性な、一般的にそれらをレンダリングする、変性させる。両方のテスト式は、アップスケーリングされ、および単離手順は、標的タンパク質の溶解度によって決定される。追加の再生またはリフォールディングステップは不溶性タンパク質のために必要である。得られた組換えタンパク質は、サイズ排除クロマトグラフィーを用いて精製することができる。 家の中での組換えタンパク質産生は、標的タンパク質のミリグラム本培養のリットルあたり単離することができるので、市販品よりもコストの利点を提供する。必要な機器のほとんどは、一般的な生物学的または化学実験室で提供されています。タンパク質工学の作成を可能に常に市販されていない追加された機能を持つカスタムの融合タンパク質の。 図1は、組換えタンパク質を工学に関わる主な手順を示している。この発現系では、我々は、インターフェロン-γ、血小板由来増殖因子、および骨形成タンパク質21〜23のような多くのbiotinylatableタンパク質を、作成しているが、我々は我々が軸索ガイダンス、NGF（29 kDaのために設計された二つのタンパク質に焦点を当てます）とのSema3A（91 kDa）の10（レビューのための基準24を参照）。ビオチン化はよく知られているビオチン-ストレプトアビジン相互作用を利用して25-27標識タンパク質の同定、単離および固定化のための一般的な技術である。生物物理学的なプローブ28,29、バイオセンサ30と、量子ドット31は、10 27 -15 MのオーダーのK dで、ビオチン-ストレプトアビジン結合の高い親和性を利用するシステムのいくつかの例である。 E.大腸菌バイオスズリガーゼ、BirAをは、ビオチンタグの付いたシーケンス18,32内で見つかったリジン側鎖へのビオチンの共有結合を助ける。材料や生体分子へのテザリングビオチンは、複数の組織工学適用21,33-35用セルに成長因子の持続性送達を生産している。したがって、これらのカスタム設計biotinylatableタンパク質を設計することは複数の研究の利益を超越することができる強力なツールです。

<table><tbody><tr><td>1,4-Dithio-DL-threitol, DTT, 99.5%</td><td>Chem-Impex International</td><td>127</td><td>100 g</td></tr><tr><td>2-Hydroxyethylmercaptan &amp;beta;-Mercaptoethanol</td><td>Chem-Impex International</td><td>642</td><td>250 ml</td></tr><tr><td>Acetic acid, glacial</td><td>EMD</td><td>AX0073-9</td><td>2.5 L</td></tr><tr><td>Agar</td><td>Bioshop</td><td>AGR001.500</td><td>500 g</td></tr><tr><td>Ampicillin sodium salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9518</td><td>25 g</td></tr><tr><td>Antifoam 204</td><td>Sigma-Aldrich</td><td>A6426</td><td>500 g</td></tr><tr><td>Barstar-NGF pET-21a(+)</td><td>GenScript USA Inc.</td><td></td><td>4 &amp;micro;g</td></tr><tr><td>BL21(DE3) Competent Cells</td><td>Novagen</td><td>69450</td><td>1 ml; Expression Host</td></tr><tr><td>Bradford reagent</td><td>Sigma-Aldrich</td><td>B6916</td><td>500 ml</td></tr><tr><td>BugBuster</td><td>Novagen</td><td>70922-3</td><td>100 ml</td></tr><tr><td>Gel filtration standard</td><td>Bio-Rad</td><td>151-1901</td><td>6 vials</td></tr><tr><td>Glycerol</td><td>Bioshop</td><td>GLY001.1</td><td>1 L</td></tr><tr><td>Guanidine hydrodioride amioformamidine hydrochloride</td><td>Chem-Impex International</td><td>152</td><td>1 kg</td></tr><tr><td>His-Pur Ni-NTA Resin</td><td>Thermo Scientific</td><td>88222</td><td>100 ml</td></tr><tr><td>Hydrochloric acid</td><td>EMD</td><td>HX0603-3</td><td>2.5 L</td></tr><tr><td>Imidazole&amp;nbsp;</td><td>Chem-Impex International</td><td>418</td><td>250 g</td></tr><tr><td>IPTG</td><td>Chem-Impex International</td><td>194</td><td>100 g</td></tr><tr><td>Laemmli sample buffer</td><td>Bio-Rad</td><td>161-0737</td><td>30 ml</td></tr><tr><td>Lauryl sulfate sodium salt, Sodium dodecyl surface</td><td>Chem-Impex International</td><td>270</td><td>500 g</td></tr><tr><td>LB Broth&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L3022</td><td>1 kg</td></tr><tr><td>NovaBlue Competent Cells</td><td>Novagen</td><td>69825</td><td>1 ml; Cloning Host</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td>10 pack</td></tr><tr><td>Potassium Chloride</td><td>Chem-Impex International</td><td>01247</td><td>1 kg</td></tr><tr><td>Sema3A-pET-21a(+)</td><td>GenScript USA Inc.</td><td></td><td>4 &amp;micro;g</td></tr><tr><td>SimplyBlue SafeStain</td><td>Invitrogen</td><td>LC6060</td><td>1 L</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S5886-1KG</td><td>1 kg</td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318-500</td><td>500 g</td></tr><tr><td>Sodium phosphate diabasic</td><td>Sigma-Aldrich</td><td>S5136-500G</td><td>500 g</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S5011</td><td>500 g</td></tr><tr><td>Terrific Broth</td><td>Bioshop</td><td>TER409.5</td><td>5 kg</td></tr><tr><td>Tetracycline hydrochloride</td><td>Chem-Impex International</td><td>667</td><td>25 g</td></tr><tr><td>Tris/Glycine/SDS Buffer, 10x</td><td>Bio-Rad</td><td>1610732</td><td>1 L</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td>1 kg</td></tr><tr><td>Tryptone, pancreatic</td><td>EMD</td><td>1.07213.1000</td><td>1 kg</td></tr><tr><td>Yeast extract, granulated</td><td>EMD</td><td>1.03753.0500</td><td>500 g</td></tr><tr><td>&amp;nbsp;&amp;Auml;KTApurifier10</td><td>GE Healthcare</td><td>28-4062-64</td><td>Includes kits and accessories</td></tr><tr><td>Benchtop Orbital Shaker</td><td>Thermo Scientific</td><td>SHKE4000</td><td>MAXQ 4000</td></tr><tr><td>BirA500</td><td>Avidity</td><td>BirA500</td><td>Enzyme comes with reaction buffers and biotin solution</td></tr><tr><td>Dialysis Casette</td><td>Thermo Scientific</td><td>66380</td><td>Slide-A-Lyzer (Extra Strength)</td></tr><tr><td>Dialysis Tubing</td><td>Spectrum Laboratories</td><td>132127, 132129</td><td>MWCO: 25,000 and 50,000</td></tr><tr><td>Flow Diversion Valve FV-923</td><td>GE Healthcare</td><td>11-0011-70</td><td></td></tr><tr><td>FluoReporter Biotin Quantification Assay Kit</td><td>Invitrogen</td><td>1094598</td><td></td></tr><tr><td>Frac-950 Tube Racks, Rack C</td><td>GE Healthcare</td><td>18-6083-13</td><td></td></tr><tr><td>Fraction Collector Frac-950</td><td>GE Healthcare</td><td>18-6083-00</td><td>Includes kits and accessories</td></tr><tr><td>Heated/Refrigerated Circulator&amp;nbsp;</td><td>VWR</td><td>13271-102</td><td>Model 1156D</td></tr><tr><td>Heating Oven FD Series</td><td>Binder</td><td></td><td>Model FD 115</td></tr><tr><td>HiLoad 16/60 Superdex 200 pg</td><td>GE Healthcare</td><td>17-1069-01</td><td>Discontinued--Replacement Product: HiLoad 16/600 Superdex 200 pg</td></tr><tr><td>J-26 XPI Avanti Centrifuge</td><td>Beckman Coulter</td><td>393126</td><td></td></tr><tr><td>JA 25.50 Rotor</td><td>Beckman Coulter</td><td>363055</td><td></td></tr><tr><td>JLA 8.1 Rotor</td><td>Beckman Coulter</td><td>969329</td><td>Includes 1 L polyporpylene bottles</td></tr><tr><td>JS 5.3 Rotor</td><td>Beckman Coulter</td><td>368690</td><td></td></tr><tr><td>Laminar Flow Hood</td><td>Themo Scientific</td><td>1849</td><td>Forma 1800 Series Clean Bench</td></tr><tr><td>Microplate Reader</td><td>TECAN</td><td></td><td>infinite M200</td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>165-8004</td><td>4-gel vertical electrophoresis system</td></tr><tr><td>Mini-PROTEAN TGX Precast Gels</td><td>Bio-Rad</td><td>456-9036</td><td>Any kDa, 15-well comb</td></tr><tr><td>Ni-NTA Column</td><td>Bio-Rad</td><td>737-2512</td><td>49 ml&amp;nbsp;volume ECONO-Column</td></tr><tr><td>Plasmid Miniprep Kit</td><td>Omega Bio-Tek</td><td>D6943-01</td><td></td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>164-5052</td><td>250 V, 3 A, 300 W</td></tr><tr><td>Round Bottom Polypropylene Copolymer Tubes</td><td>VWR</td><td>3119-0050</td><td>50 ml&amp;nbsp;tubes for JA 25.50 rotor</td></tr><tr><td>Spin-X UF Concentrators</td><td>Corning</td><td>431488, 431483</td><td>&amp;nbsp;20 and 6 ml; MWCO: 10,000 Da</td></tr><tr><td>Subcloning Service</td><td>GenScript USA Inc.</td><td></td><td>Protein Services</td></tr><tr><td>Ultrasonic Processor&amp;nbsp;</td><td>Cole-Parmer</td><td>18910445A</td><td>Model CV18</td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>SI-0236</td><td>Model G560</td></tr></tbody></table>

expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration

組換えタンパク質工学は、 大腸菌（E.coli）発現ほぼ40年のためのシステム、今日Eを利用している大腸菌はまだ最も広く使用されている宿主生物である。システムの柔軟性は、（ストレプトアビジン相互作用のための）ビオチンタグなどの部分と緑色蛍光タンパク質またはチェリーレッドのタンパク質のような大きな機能性タンパク質を付加することができます。また、翻訳後修飾を付与する金属イオンキレート剤、一意的に反応性官能基、分光学的プローブ、および分子などの非天然アミノ酸の組込みは、タンパク質の特性及び機能のより良好な操作を可能にした。その結果、この技術は研究の様々な分野のための重要なユーティリティを提供し、カスタマイズ可能な融合タンパク質を作成します。具体的には、biotinylatableタンパク質配列が原因アビジンおよびストレプトアビジンとビオチンとの間の高親和性相互作用の多くの標的タンパク質に組み込まれている。この追加により、タグ付けされたタンパク質の検出および精製を強化するだけでなく、このような細胞選別などの二次アプリケーションのための道を開くことに支援しています。このように、ビオチン標識分子がbioindustrialおよび生物医学分野で増加し、広範な影響力を示している。本研究の目的のために、我々は、神経成長因子（NGF）およびsemaphorin3A（Sema3Aの）機​​能的領域を含む組換えビオチン化融合タンパク質を設計している。我々は、これらのビオチン化融合タンパク質は、他の活性なタンパク質配列と共に、組織工学および再生の目的のための生体材料に繋留することができる方法を以前に報告した。このプロトコルは、T7 LAC誘導ベクターおよびEを利用し、ミリグラム規模でエンジニアリングbiotinylatableタンパク質の基礎を概説形質転換からのスケールアップおよび精製を開始大腸菌発現宿主、。

<html> クローニングとテスト式 めっきが適切に行われると、単一の単離されたコロニーをクローン形質転換された細菌細胞（ 図2A）プラッキングの可能性を高めるために形成すべきである。あまりにも多くの細胞を播種する場合は、プレートを37℃以下で変態が長すぎるインキュベートし、コロニーを寒天プレートを被覆または細胞の大きな凝集?...

Watch this Scientific Journal Video about Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration at JoVE.com

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

biotinylatable融合タンパク質を開発することは、研究の様々な分野で多くの潜在的な用途がある。組換えタンパク質工学は、カスタム設計されたタンパク質を高収率で提供する、費用対効果のある単純な手順である。

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 ...

expression-isolation-purification-soluble-insoluble-biotinylated

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

33.5K ビュー.  University of Akron.  biotinylatable融合タンパク質を開発することは、研究の様々な分野で多くの潜在的な用途がある。組換えタンパク質工学は、カスタム設計されたタンパク質を高収率で提供する、費用対効果のある単純な手順である。 

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

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

ビオチン化細胞表面タンパク質の精製<em&gt; Rhipicephalus microplus</em&gt;上皮腸細胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

生化学

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

ビオチン標識を用いてタンパク質核酸相互作用を研究するインビトロ生化学アッセイ

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

遺伝学

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

神経科学

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

一次ニューロンにおける量子ドットで標識されたBDNFの軸索輸送のリアルタイムイメージング

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

神経化学的マーカーのためのビオシチンで満たされたと加工セクションの免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

ビオチン化を用いた初代星状細胞培養におけるタンパク質の細胞表面発現およびエンドサイトーシス速度の測定

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

体外における感覚神経と運動神経からの線維芽細胞およびシュワン細胞の精製

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

ニューロンの細胞密売研究のためのチューブ内の組換えBDNFを精製し、直接モノビオチン化するための改良されたプロトコル

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

細胞表面ビオチン化アッセイ

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

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

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ビオチン化細胞表面タンパク質の精製