This research was funded by the Office of Naval Research through Core funds provided to the Naval Research Laboratory.

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الكتاب ليس لديهم ما يكشف.

يعمل هذا البروتوكول على إثبات وجود ممثل الموجهة تقنية التعبئة والتغليف التي تنتج انزيم من الفائدة وتعبئتها في OMV من قبل كولاي. كما هو الحال مع العديد من التقنيات المعقدة هناك مناطق متعددة في أي بروتوكول يمكن تعديلها لاستيعاب لاستخدامها في تطبيقات فريدة من نوعها...

المقدمة هنا هي طريقة لتصميم وإنتاج وتنقية البكتيرية حويصلات الغشاء الخارجي محملة انزيم (OMV). OMV صغيرة، unilamellar في المقام الأول، proteoliposomes التي تتراوح في حجمها 30-200 نانومتر 1،2. وقد أثبتت جميع البكتيريا سالبة الجرام وموجبة الجرام التي تم دراستها لتاريخ الافراج عنه إما OMV أو الحويصلات خارج الخلية (EV) من سطحها 3،4. آلية دقيقة التي يتم إنتاجها OMV لم يتم بعد توضح تماما بسبب التجمعات البكتيرية المتنوعة التي تفرز لهم، وكذلك الوظائف المختلفة التي تخدمها. وقد ثبت OMV لنقل مجموعة واسعة من البضائع من الجزيئات الصغيرة، والببتيدات والبروتينات والمادة الوراثية تقدم مجموعة متنوعة من الإشارات المعقدة، النبات الجينات، والفوعة تقوم بعملها 5،6. الآليات الدقيقة لOMV نشوء حيوي لا تتميز بشكل جيد، ويبدو أن تختلف بين الأنواع البكتيرية. وعلى الرغم من ثيالصورة الحقيقة، قمنا بتطوير طريقة لتعزيز كفاءة التعبئة والتغليف من البروتين المؤتلف في OMV عن طريق إنشاء الربط الاصطناعية بين بروتين من الفائدة والبروتين الذاتية وفيرة للغاية لالغشاء الخارجي للبكتيريا ولاحق OMV. في حالة عدم وجود الربط الاصطناعية، أو تقارب إدراجها بشكل مصطنع، بين البروتين المعبر عنه recombinantly وOMV كفاءة التعبئة والتغليف المرصودة منخفضة جدا (7). هذه النتيجة هي التي يمكن توقعها على تضمين البروتينات داخل OMV يحدث إما عن طريق تغليف فرصة عشوائي في نفس اللحظة من تشكيل OMV على سطح البكتيريا أو من خلال التعبئة والتغليف إخراج الآليات التي ليست مفهومة جيدا. وقد لوحظ بعض النجاح في التعبئة والتغليف البروتينات ببساطة من خلال أكثر من التعبير في الفضاء محيط بالجبلة التي تعتمد على تغليف فرصة عشوائي ولكن التعبئة الفعالة هي عالية البروتين تعتمد مع بعض البروتينات التعبئة والتغليف في كفاءة عالية بالمقارنة مع الآخرين عشرفي لا حزمة في كل 8-10. من خلال استخدام تقنيات البيولوجيا التركيبية المشتركة سعينا لهندسة الإشريكية القولونية (إي كولاي) لإنتاج واحد، حزمة وتفرز انزيم نشط من الاهتمام الى OMV أن تلتف القيود المعرفة الحالية بشأن كيف تتشكل OMV وكيف يتم تحديد البضائع عن طريق البكتيريا ل التعبئة والتغليف. لأغراض هذا التطبيق تم اختيار نظام البروتين bioconjugation الانقسام كما الربط الاصطناعية من خيار لتسهيل التعبئة والتغليف الاتجاه إلى OMV. وكما يوحي اسمها يتكون نظام البروتين bioconjugation تقسيم اثنين من المجالات فرعية التكميلية التي تتفاعل مع بعضها البعض. ويشار إلى المجالات البروتين الانقسام التي تم اختيارها لأغراض هذا البروتوكول باسم SpyCatcher (SC) وSpyTag (ST) المجال و هي مستمدة من المكورات العقدية المقيحة ملزمة فبرونيكتين البروتين (FbaB) 11. هذا النظام بروتين الانقسام هو غير عادي في أن ثالدجاجة مفارز هما على مقربة سندات isopeptide تشكل بشكل عفوي بين الداني الأسبارتيك بقايا حمض أميني ويسين خلق صلة التساهمية. لا يتطلب تشكيل السندات Isopeptide إضافة البروتينات كوصي، والإنزيمات المحفزة، أو العوامل المساعدة ويمكن أن يحدث بسهولة في درجة حرارة الغرفة (RT) وأكثر من مجموعة واسعة من الشروط ذات الصلة من الناحية الفسيولوجية 12. كدليل على المفهوم، وقد تم اختيار phosphotriesterase (PTE) (EC 3.1.8.1) من Brevundimonas الضئيلة ليتم تعبئتها في كولاي المستمدة OMV 13. يحتوي PTE على ثنائي النواة الموقع الزنك / الزنك نشط ولديه القدرة على تحطيم الفوسفات العضوية من خلال رد فعل التحلل تحويل aryldialkylphosphates إلى dialkylphosphates والكحول أريل 14. التعرض لالفوسفات العضوية يضعف وظيفة النواقل العصبية السليمة من خلال تثبيط المائي للأستيل كولين قبل أستيل عند التقاطعات العصبية والعضلية ماكينز الفوسفات العضوي المركبات المشتقة خطيرة للغاية (15). لفترات طويلة أو التعرض الكبير لالفوسفات العضوية ينتج عادة في التشنجات لا يمكن السيطرة عليها، وعادة ما يسبب الموت عن طريق الاختناق. في حين PTE يسلك أعلى نشاط الحفاز نحو الباراأكسيون، مبيد حشري قوية جدا، بل هو أيضا قادرة على hydrolyzing مجموعة واسعة من المبيدات الأخرى وV / G نوع غاز الأعصاب الكيميائي 16. لتسهيل OMV التعبئة والتغليف، وقد تم تصميم البلازميد البكتيري أن يشفر بناء الجين الذي يحتوي على المروج محرض، سلسلة توطين محيط بالجبلة، ويبعد مسافة قصيرة متعددة موقع استنساخ المنبع من سلسلة SC الجينات. غرز الجينات PTE بين القائد وتسلسل SC يسمح لخلق التحول الجيني الذي يستهدف تدريب المدرسين-SC الانصهار البروتين إلى الفضاء محيط بالجبلة لOMV التعبئة والتغليف. في حين أن جهود وصفها هنا تركز على تدريب المدرسين، وجين الإنزيم قابلة للتبديل، ويمكن بسهولة أن يتم استبدال مع تسلسل الجينات آخر لبناء واجهاتالتعبئة والتغليف ilitate إنزيم بديل أو البروتين. وبما أن الجزء الثاني من الربط الاصطناعية، يتم اختيار وفيرة الخارجي بروتين الغشاء (OmpA) لتقديم تسلسل الببتيد ST. في حين أن اختيار البروتين مرساة يمكن أن تختلف، فمن الضروري أن البروتين يحتوي على نطاق المتساهل الذي يطرح داخل الفضاء محيط بالجبلة، تسامح بناء الانصهار دون إحداث السمية الخلوية، ومن المعروف أن تكون موجودة في OMV، ولا تجميع عندما يكون recombinantly أنتجت. OmpA هو 37.2 كيلو دالتون بروتين الغشاء PORIN أن يعرف أن يتم التعبير عنها بشكل كبير في الغشاء الخارجي للبكتيريا ولاحق OMV 17. هو متورط في نقل الجزيئات الصغيرة، &lt;2 نانومتر في الحجم، عبر غشاء البكتيريا (18). مواطن OmpA اثنين من المجالات فريدة من نوعها من الناحية الهيكلية، والغشاء بيتا برميل عزر وجزء للذوبان periplasmically C-محطة معروفة للتفاعل مع ببتيدوغليكان 19. في متحولة OmpA-ST الانصهار المصممد هنا تم حذف الجزء C-محطة من OmpA وتنصهر في ST إلى N- التي تواجه periplasmically أو C-ترميني. حذف جزء محيط بالجبلة من OmpA يقلل من عدد من التفاعلات بين الغشاء الخارجي وببتيدوغليكان مما أدى إلى زعزعة استقرار الغشاء مما يؤدي إلى فرط تحوصل 7. واستمر الجينوم OmpA بالإضافة إلى بناء OmpA-ST أعرب recombinantly للتخفيف الإجمالي زعزعة استقرار الغشاء.

<table><tbody><tr><td>IPTG</td><td>Any</td><td></td><td>Always prepare fresh or aliquot and freeze.</td></tr><tr><td>L-arabinose</td><td>Any</td><td></td><td>Can be prepared ahead of time and stored at 4 &amp;deg;C.</td></tr><tr><td>Ampicillin</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>TB/LB Culture Media</td><td>Any</td><td></td><td>Other growth medias will likely work similarly.</td></tr><tr><td>Triton X-100</td><td>Any</td><td></td><td>One of many potential suitable surfactants.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Baffled culture flasks</td><td>Any</td><td></td><td>The baffles promote higher levels of aeration.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CHES</td><td>Fisher Bioreagents</td><td>BP318-100</td><td>Optimal buffer used for paraoxon degredation (pH &gt; 8).</td></tr><tr><td>Paraoxon</td><td>Chem Service</td><td>N-12816</td><td>Very toxic substance to be handled carefully and disposed of properly.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Syringe Filter 0.45 &amp;micro;m</td><td>Thermo Scientific</td><td>60183-221 (30 mm)</td><td>Filter diameter will depend on volume of sample. Low protein binding membrane is critical.</td></tr><tr><td>Shaker incubator</td><td>New Brunswick</td><td>Excella E24</td><td>Precise temperature and mixing is essential for reproducable bacterial growth.&amp;nbsp;</td></tr><tr><td>Sorvall Culture Centrifuge</td><td>Thermo Scientific</td><td>RC 5B PLUS</td><td>Large volume (500 ml) culture centrifuge capable of 7,000 x g.</td></tr><tr><td>Sorvall Ultracentrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>WX Ultra 90</td><td>Capable of centrifugal forces &amp;ge;150,000 x g.</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Thermo Scientific</td><td>AH-629</td><td>Ensure the proper rotor and tubes are used and that everything is properly balanced.</td></tr><tr><td>Ultra-Clear Ultracentrifuge Tubes (25 x 89 mm)</td><td>Beckman Coulter</td><td>344058</td><td>Ensure no stress fractures are present prior to use and that tubes are presicely balanced.</td></tr><tr><td>Spectrophotometer&amp;nbsp;</td><td>Tecan</td><td>Infinite M1000</td><td>Necessary for enzyme kinetic assays.</td></tr><tr><td>DLS/particle tracking</td><td>NanoSight&amp;nbsp;</td><td>LM10</td><td>Necessary for OMV size distribution and concentration determination.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>BL21(DE3)</td><td>NEB</td><td></td><td>Suitable bacterial expression strain.</td></tr><tr><td>pET22</td><td>EMD Millipore</td><td>69744-3</td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>pACYC184</td><td>NEB</td><td></td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Example kit.</td></tr></tbody></table>

directed protein packaging within outer membrane vesicles from escherichia coli: design, production and purification

اهتمام متزايد في تطبيق تقنيات البيولوجيا الاصطناعية لبرنامج حويصلات الغشاء الخارجي (OMV) تؤدي إلى بعض التطبيقات مثيرة جدا للاهتمام وفريدة من نوعها لOMV حيث النانوية التقليدية تثبت من الصعب جدا لتجميع. حتى الآن، وقد ثبت عن البكتيريا سالبة الجرام لانتاج أو.ام.في التظاهر التعبئة والتغليف من مجموعة متنوعة من البضائع التي تتضمن جزيئات صغيرة، والببتيدات والبروتينات والمادة الوراثية. وبناء على حمولتها متنوعة، وتورط OMV في العديد من العمليات البيولوجية التي تتراوح بين الاتصالات خلية خلية لنقل الجينات وتسليم عوامل الفوعة اعتمادا عليها البكتيريا تنتج أو.ام.في. إلا في الآونة الأخيرة البكتيرية OMV تكون ميسرة للاستخدام عبر مجموعة واسعة من التطبيقات من خلال تطوير تقنيات لمراقبة والتعبئة والتغليف مباشرة من البروتينات المؤتلف في OMV. يصف هذا البروتوكول وسيلة لإنتاج وتنقية، واستخدام انزيم تعبئتها OMV توفير لتحسين overaإنتاج ليرة لبنانية من انزيم المؤتلف، وزيادة البثور، وتعزيز الاستقرار الانزيم. واستخدام الناجح لهذا البروتوكول يؤدي إلى خلق السلالة البكتيرية التي تنتج في وقت واحد بروتين المؤتلف، وتوجهها لOMV التغليف من خلال استحداث رابط الاصطناعية بين البروتين المؤتلف والخارجي البروتين غشاء مرساة. تفاصيل هذا البروتوكول أيضا طرق لعزل OMV من الثقافات البكتيرية وكذلك التقنيات السليمة للتعامل معها والأشياء في الاعتبار عند التكيف مع هذا البروتوكول للاستخدام في تطبيقات أخرى فريدة من نوعها مثل: تسليم الصيدلانية المخدرات والتشخيص الطبي، وإصلاح البيئة.

التعبير في وقت واحد من اثنين من البروتينات المؤتلف، كما هو مطلوب لاستراتيجية التعبئة والتغليف OMV بالتفصيل في هذا البروتوكول، ويمكن تحقيق ذلك من خلال عدد من السبل المختلفة. هنا، تم استخدام نظام اثنين من ناقلات مع أصول المتوافقة مع النسخ المتماثل وأش...

Watch this Scientific Journal Video about Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification at JoVE.com

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

توجه التغليف البروتين داخل الخارجي غشاء الحويصلات من<em&gt; كولاي</em&gt;: تصميم وإنتاج وتنقية

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

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Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

13.3K Views.  Naval Research Laboratory. A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented. 

Video: Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

إنتاج وبلورة وتحديد هيكل<em&gt; C. صعب</em&gt; PPEP-1 عن طريق Microseeding والزنك والصحراء

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

من التركيبات لبلورات - نحو تحديد هيكل β برميل الخارجي بروتينات الغشاء

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

الأسلوب كفاءة ريفولدينج وتنقية ليجند تشيموريسيبتور ملزمة المجال

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

توصيف الناقلات غشاء بواسطة تعبير مغاير في كولاي وإنتاج الحويصلات الغشائية

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

تصميم وبناء الاصطناعي خارج الخلية مصفوفة (aECM) والبروتينات من<em&gt; الإشريكية القولونية</em&gt; للبشرة هندسة الأنسجة

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Bioengineering

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

الإشريكيّة القولونية-"على أساس تخليق البروتين" الخلية الحرة: البروتوكولات لتكنولوجيا منصة قوية ومرنة، وموجودا

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

توجيه تجميع البروتينات الشبيهة بالإيلاستين إلى هياكل فوق جزيئية محددة وتغليف البضائع في المختبر

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

تحديد بروتين في مجمعات<em&gt; كولاي</em&gt; استخدام المتعاقبة تنقية تقارب الببتيد في تركيبة مع جنبا إلى جنب الطيف الكتلي

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Biology

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

الإنتاج والتصور جدارها البكتيرية وبروتوبلاستس لتميز التعريب الببتيد مضادات الميكروبات

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

عزل وتنقية الحويصلات خارج الخلية من الإشريكية القولونية والبكتيريا الأخرى قابلة للتطوير

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

توجه التغليف البروتين داخل الخارجي غشاء الحويصلات من

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