<meta charset="utf8"></head><body>利 什曼原虫 翻译提取物 （LTE） 生产的简化方案

<ol>
	<li>Nirenberg, M. W., Matthaei, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dependence+of+cell-free+protein+synthesis+in+E.coli+upon+naturally+occurring+or+synthetic+polyribonucleotides.">The dependence of cell-free protein synthesis in E.coli upon naturally occurring or synthetic polyribonucleotides.</a> Proc Natl Acad Sci USA. 47 (10), 1588-1602 (1961).</li><li>Caschera, F., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+2.3+mg/ml+of+protein+with+an+all+Escherichia+coli+cell-free+transcription-translation+system.">Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system.</a> Biochimie. 99, 162-168 (2014).</li><li>Kelwick, R., Webb, A. J., MacDonald, J. T., Freemont, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Bacillus+subtilis+cell-free+transcription-translation+system+for+prototyping+regulatory+elements.">Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.</a> Metab Eng. 38, 370-381 (2016).</li><li>Ezure, 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=Cell-free+protein+synthesis+system+prepared+from+insect+cells+by+freeze-thawing.">Cell-free protein synthesis system prepared from insect cells by freeze-thawing.</a> Biotechnol Prog. 22 (6), 1570-1577 (2006).</li><li>Harbers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheat+germ+systems+for+cell-free+protein+expression.">Wheat germ systems for cell-free protein expression.</a> FEBS Letters. 588 (17), 2762-2773 (2014).</li><li>Kobs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+the+cell-free+protein+expression+system+that+meets+your+experimental+goals.">Selecting the cell-free protein expression system that meets your experimental goals.</a> Promega Corporation. 21, 6-9 (2008).</li><li>Kovtun, O., 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=Leishmania+cell-free+protein+expression+system.">Leishmania cell-free protein expression system.</a> Methods. 55 (1), 58-64 (2011).</li><li>Basile, G., Peticca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Leishmania+tarentolae.">Recombinant protein expression in Leishmania tarentolae.</a> Mol Biotechnol. 43 (3), 273-278 (2009).</li><li>Mureev, S., Kovtun, O., Nguyen, U. T., Alexandrov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-independent+translational+leaders+facilitate+cell-free+expression.">Species-independent translational leaders facilitate cell-free expression.</a> Nat Biotechnol. 27 (8), 747-752 (2009).</li><li>Gambin, Y., 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=Single-molecule+fluorescence+reveals+the+oligomerization+and+folding+steps+driving+the+prion-like+behavior+of+ASC.">Single-molecule fluorescence reveals the oligomerization and folding steps driving the prion-like behavior of ASC.</a> J Mol Biol. 430 (4), 491-508 (2018).</li><li>Sierecki, E., 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=Rapid+mapping+of+interactions+between+human+SNX-BAR+proteins+measured+in+vitro+by+AlphaScreen+and+single-molecule+spectroscopy.">Rapid mapping of interactions between human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy.</a> Mol Cell Proteomics. 13 (9), 2233-2245 (2014).</li><li>Sierecki, E., 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=Nanomolar+oligomerization+and+selective+co-aggregation+of+alpha-synuclein+pathogenic+mutants+revealed+by+single-molecule+fluorescence.">Nanomolar oligomerization and selective co-aggregation of alpha-synuclein pathogenic mutants revealed by single-molecule fluorescence.</a> Sci Rep. 6, 37630 (2016).</li><li>Leitao, A., Bhumkar, A., Hunter, D. J. B., Gambin, Y., Sierecki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+a+selective+mechanism+for+the+inhibition+of+alpha-synuclein+aggregation+by+beta-synuclein.">Unveiling a selective mechanism for the inhibition of alpha-synuclein aggregation by beta-synuclein.</a> Int J Mol Sci. 19 (2), 334 (2018).</li><li>Gambin, Y., 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=Single-molecule+analysis+reveals+self+assembly+and+nanoscale+segregation+of+two+distinct+cavin+subcomplexes+on+caveolae.">Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae.</a> Elife. 3, e01434 (2013).</li><li>Ve, 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=Structural+basis+of+TIR-domain-assembly+formation+in+MAL-+and+MyD88-dependent+TLR4+signaling.">Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling.</a> Nat Struct Mol Biol. 24 (9), 743-751 (2017).</li><li>Guo, 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=Subunit+organisation+of+in+vitro+reconstituted+HOPS+and+CORVET+multisubunit+membrane+tethering+complexes.">Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes.</a> PLoS One. 8 (12), e81534 (2013).</li><li>Ruehrer, S., Michel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+Leishmania+tarentolae+cell-free+extracts+for+the+synthesis+of+human+solute+carriers.">Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers.</a> Mol Membr Biol. 30 (4), 288-302 (2013).</li><li>Varasteh Moradi, 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=Mapping+Interactions+among+cell-free+expressed+Zika+virus+proteins.">Mapping Interactions among cell-free expressed Zika virus proteins.</a> J Proteome Res. 19 (4), 1522-1532 (2020).</li><li>Gagoski, D., 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=Cell-free+pipeline+for+discovery+of+thermotolerant+xylanases+and+endo-1,4-beta-glucanases.">Cell-free pipeline for discovery of thermotolerant xylanases and endo-1,4-beta-glucanases.</a> J Biotechnol. 259, 191-198 (2017).</li><li>Ergun Ayva, 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=Exploring+performance+parameters+of+artificial+allosteric+protein+switches.">Exploring performance parameters of artificial allosteric protein switches.</a> J Mol Biol. 434 (17), 167678 (2022).</li><li>Lau, D., 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=Fluorescence+biosensor+for+real-time+interaction+dynamics+of+host+proteins+with+HIV-1+capsid+tubes.">Fluorescence biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes.</a> ACS Appl Mater Interfaces. 11 (38), 34586-34594 (2019).</li><li>Ryan, S. M., 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=Novel+antiinflammatory+biologics+shaped+by+parasite-host+coevolution.">Novel antiinflammatory biologics shaped by parasite-host coevolution.</a> Proc Natl Acad Sci USA. 119 (36), e2202795119 (2022).</li><li>McMahon, K. A., 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=Identification+of+intracellular+cavin+target+proteins+reveals+cavin-PP1alpha+interactions+regulate+apoptosis.">Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis.</a> Nat Commun. 10 (1), 3279 (2019).</li><li>Sierecki, E., 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=A+cell-free+approach+to+accelerate+the+study+of+protein-protein+interactions+in+vitro.">A cell-free approach to accelerate the study of protein-protein interactions in vitro.</a> Interface Focus. 3 (5), 20230018 (2013).</li><li>Johnston, W. A., Moradi, S. V., Alexandrov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaption+of+the+Leishmania+cell-free+expression+system+to+high-throughput+analysis+of+protein+interactions.">Adaption of the Leishmania cell-free expression system to high-throughput analysis of protein interactions.</a> Methods Mol Biol. 2025, 403-421 (2019).</li><li>Jung, W., 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=Cell-free+formation+and+interactome+analysis+of+caveolae.">Cell-free formation and interactome analysis of caveolae.</a> J Cell Biol. 217 (6), 2141-2165 (2018).</li><li>Fontaine, F. R., 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=Functional+domain+analysis+of+SOX18+transcription+factor+using+a+single-chain+variable+fragment-based+approach.">Functional domain analysis of SOX18 transcription factor using a single-chain variable fragment-based approach.</a> MAbs. 10 (4), 596-606 (2018).</li><li>Overman, J., 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=Pharmacological+targeting+of+the+transcription+factor+SOX18+delays+breast+cancer+in+mice.">Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.</a> Elife. 6, e21221 (2017).</li><li>Kubala, M. H., 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=Mammalian+farnesyltransferase+alpha+subunit+regulates+vacuolar+protein+sorting-associated+protein+4A+(Vps4A)--dependent+intracellular+trafficking+through+recycling+endosomes.">Mammalian farnesyltransferase alpha subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.</a> Biochem Biophys Res Commun. 468 (4), 580-586 (2015).</li><li>Han, S. P., 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=Cortactin+scaffolds+Arp2/3+and+WAVE2+at+the+epithelial+zonula+adherens.">Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.</a> J Biol Chem. 289 (11), 7764-7775 (2014).</li><li>Das Gupta, 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=Class+IIa+histone+deacetylases+drive+toll-like+receptor-inducible+glycolysis+and+macrophage+inflammatory+responses+via+pyruvate+kinase+M2.">Class IIa histone deacetylases drive toll-like receptor-inducible glycolysis and macrophage inflammatory responses via pyruvate kinase M2.</a> Cell Rep. 30 (8), 2712-2728.e8 (2020).</li><li>Leit&#227;o, A. D. G., 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=Selectivity+of+protein+interactions+along+the+aggregation+pathway+of+&#945;-synuclein.">Selectivity of protein interactions along the aggregation pathway of &#945;-synuclein.</a> BioRxiv. , (2021).</li><li>Gagoski, D., 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=Performance+benchmarking+of+four+cell-free+protein+expression+systems.">Performance benchmarking of four cell-free protein expression systems.</a> Biotechnol Bioeng. 113 (2), 292-300 (2016).</li><li>Johnston, W. A., Alexandrov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+eukaryotic+cell-free+lysate+from+Leishmania+tarentolae.">Production of eukaryotic cell-free lysate from Leishmania tarentolae.</a> Methods Mol Biol. 1118, 1-15 (2014).</li><li>Hunter, D. J. B., Bhumkar, A., Giles, N., Sierecki, E., Gambin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+instabilities+explain+batch-to-batch+variability+in+cell-free+protein+expression+systems.">Unexpected instabilities explain batch-to-batch variability in cell-free protein expression systems.</a> Biotechnol Bioeng. 115 (8), 1904-1914 (2018).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >PD-10 SuperDex 25 Columns</td>
			<td >Cytiva</td>
			<td >17085101</td>
			<td >Gel filtration columns</td>
		</tr>
		<tr >
			<td >Nitrogen Cavitation cell disrupter</td>
			<td >Parr Industries</td>
			<td >4635 or 4639</td>
			<td>Cell Disrupter</td>
		</tr>
		<tr >
			<td >Bovine derived Hemin</td>
			<td >Sigma-Aldrich</td>
			<td >H5533</td>
			<td>Culture additive</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin 10000U/ml</td>
			<td >Thermo-Fisher</td>
			<td >15140122</td>
			<td>Antibiotic mix</td>
		</tr>
		<tr >
			<td >Optiplate 384</td>
			<td >Perkin-Elmer</td>
			<td >6007290</td>
			<td>Multiwell plate for 10ul expressions</td>
		</tr>
		<tr >
			<td >Oligonucleotide</td>
			<td >IDT synthesis</td>
			<td ></td>
			<td>&#160;Oligo with sequence CAATAAAGTACAGAAACTGATAC TTATATAGCGTT</td>
		</tr>
		<tr >
			<td >Creatine Phosphokinase</td>
			<td >Sigma-Aldrich</td>
			<td >9001-15-4</td>
			<td>Enzyme</td>
		</tr>
		<tr >
			<td >Tecan Spark</td>
			<td >Tecan</td>
			<td ></td>
			<td >or similar Multimode Platereader</td>
		</tr>
		<tr >
			<td >Chemidoc MP Imager</td>
			<td >Biorad</td>
			<td ></td>
			<td>or similar SDS-PAGE gel Imager</td>
		</tr>
		<tr >
			<td >4-12% Bis-Tris Gels</td>
			<td >Invitrogen</td>
			<td >NW04125</td>
			<td>SDS-PAGE gels</td>
		</tr>
		<tr >
			<td >Biophotometer</td>
			<td >Eppendorf</td>
			<td ></td>
			<td >or similar Cuvette Specrophotometer</td>
		</tr>
		<tr >
			<td >Nanodrop One</td>
			<td >Thermofisher</td>
			<td ></td>
			<td>Nanodrop spectrophotometer</td>
		</tr>
		<tr >
			<td >Avanti JXN-26 centrifuge</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td>or similar centrifuge, with rotors/tubes rated 10K and 50K g</td>
		</tr>
		<tr >
			<td >5424R microcentrifuge</td>
			<td >Eppendorf</td>
			<td ></td>
			<td>or similar microcentrifuge, with 1.5ml microcentrifuge tubes</td>
		</tr>
		<tr >
			<td >Flask Incubator Inova S44i</td>
			<td >Eppendorf</td>
			<td ></td>
			<td>or similar flask incubator shaker suitable for 5L Flasks</td>
		</tr>
		<tr >
			<td >5L glass culture flasks</td>
			<td ></td>
			<td ></td>
			<td>Baffled glass flasks for culture growth</td>
		</tr>
		<tr >
			<td >Bactotryptone</td>
			<td >BD</td>
			<td >211705</td>
			<td>Growth medium</td>
		</tr>
		<tr >
			<td >Yeast Extract</td>
			<td >Merck</td>
			<td >VM930053</td>
			<td>Growth medium</td>
		</tr>
		<tr >
			<td >Glycerol</td>
			<td ></td>
			<td ></td>
			<td>Any analytical grade</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td ></td>
			<td ></td>
			<td>Any analytical grade</td>
		</tr>
		<tr >
			<td >KH2PO4</td>
			<td ></td>
			<td ></td>
			<td>Any analytical grade</td>
		</tr>
		<tr >
			<td >K2HPO4</td>
			<td ></td>
			<td ></td>
			<td>Any analytical grade</td>
		</tr>
		<tr >
			<td >UltraPure water</td>
			<td >Invitrogen</td>
			<td >10977-015</td>
			<td>Or output from any MilliQ-type water dispenser</td>
		</tr>
	</tbody>
</table>

production and optimization of lte, a leishmania tarentolae derived cell-free protein expression system for recombinant protein production

<meta charset="utf8"></head><body>作者聚焦：使用 LTE 真核生物无细胞蛋白表达系统生产蛋白质

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

production-optimization-lte-leishmania-tarentolae-derived-cell-free

Without Section

Research

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

JoVE Journal

Bioengineering

510 次观看.  Queensland University of Technology. 我们的无细胞蛋白表达系统通常需要冷链进行运输，现在有了开创性的冻干版本。此版本在环境温度下保持稳定，使其成为第一个以这种形式提供的真核生物系统。这简化了到实验室和现场应用的运输。目前无细胞蛋白生产的实验挑战包括优化反应条件以确保重组蛋白正确折叠和活化。尽管与传统体内蛋白表达相比，无细胞技术的成本效益仍然...

视频: 作者聚焦：使用 LTE 真核生物无细胞蛋白表达系统生产蛋白质

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

科研

生物工程

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

快速生长的纳氏弧菌的无细胞蛋白表达

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

使用无细胞自动诱导工作流程在24小时内从细胞到无细胞蛋白质合成

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

膜蛋白在巨型单层囊泡中的无细胞表达

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

作者聚焦：合成细胞中的膜蛋白重构

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

LTE 的生产和优化，一种用于重组蛋白生产的利 什曼原虫 衍生的无细胞蛋白表达系统

LTE 的生产和优化，一种用于重组蛋白生产的利什曼原虫衍生的无细胞蛋白表达系统