Name: homogeneous glycoconjugate produced by combined unnatural amino acid incorporation and click-chemistry for vaccine purposes
Uploaded: 2020-12-19T13:00:00
Description: Watch this Scientific Journal Video about Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes at JoVE.com

E.C感谢卢瓦尔支付协会（Pari科学项目"BioSynProt"）的财政支持，特别是向T.V.提供博士学位。我们还感谢Robert B. Quast博士（INRA UMR0792，CNRS UMR5504，LISBP，法国图卢兹）的宝贵技术建议。

1. UAA的合成：丙丙基-莱辛（PrK）
<ol><li>N α- Boc - propargyl -lysine的合成 18<ol>
<li>将 500 毫克的 Boc-L-Lys-OH（2.03 mmol）溶解在烧瓶中，将水性 1 M NaOH （5 mL） 和 THF （5 mL） 混合在烧瓶中，将烧瓶与硅隔膜混合。</li>
		<li>在冰浴中冷却烧瓶，然后在搅拌时使用微锡林格滴（2-3 分钟）滴滴添加 158 μL 的丙丙基氯仿酸（1.62 mmol）。</li>
		<li>将反应混合物加热至室温，继续搅?...

<ol>
	<li>Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoconjugate+vaccines:+Principles+and+mechanisms.">Glycoconjugate vaccines: Principles and mechanisms.</a> Science Translational Medicine. 10 (456), (2018).</li><li>Verez-Bencomo, V., 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+synthetic+conjugate+polysaccharide+vaccine+against+Haemophilus+influenzae+type+b.">A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b.</a> Science (New York, N.Y). 305 (5683), 522-525 (2004).</li><li>Berti, F., Adamo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+glycoconjugate+vaccines:+an+overview+of+classic+and+modern+approaches+for+protein+modification.">Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification.</a> Chemical Society Reviews. 47 (24), 9015-9025 (2018).</li><li>Stefanetti, 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=Sugar-Protein+Connectivity+Impacts+on+the+Immunogenicity+of+Site-Selective+Salmonella+O-Antigen+Glycoconjugate+Vaccines.">Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines.</a> Angewandte Chemie (International Ed. in English). 54 (45), 13198-13203 (2015).</li><li>Kay, E., Cuccui, J., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+production+of+recombinant+glycoconjugate+vaccines.">Recent advances in the production of recombinant glycoconjugate vaccines.</a> NPJ Vaccines. 4, 16 (2019).</li><li>Ma, Z., Zhang, H., Wang, P. G., Liu, X. W., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+adjacent+to+glycosylation+sites+impacts+immunogenicity+of+glycoconjugate+vaccine.">Peptide adjacent to glycosylation sites impacts immunogenicity of glycoconjugate vaccine.</a> Oncotarget. 9 (1), 75-82 (2018).</li><li>Grayson, E. J., Bernardes, G. J. L., Chalker, J. M., Boutureira, O., Koeppe, J. R., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coordinated+synthesis+and+conjugation+strategy+for+the+preparation+of+homogeneous+glycoconjugate+vaccine+candidates.">A coordinated synthesis and conjugation strategy for the preparation of homogeneous glycoconjugate vaccine candidates.</a> Angewandte Chemie (International Ed. in English). 50 (18), 4127-4132 (2011).</li><li>Pillot, 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=Site-Specific+Conjugation+for+Fully+Controlled+Glycoconjugate+Vaccine+Preparation.">Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation.</a> Frontiers in Chemistry. , (2019).</li><li>Neumann-Staubitz, P., Neumann, 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+use+of+unnatural+amino+acids+to+study+and+engineer+protein+function.">The use of unnatural amino acids to study and engineer protein function.</a> Current Opinion in Structural Biology. 38, 119-128 (2016).</li><li>Dumas, A., Lercher, L., Spicer, C. D., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+logical+codon+reassignment+-+Expanding+the+chemistry+in+biology.">Designing logical codon reassignment - Expanding the chemistry in biology.</a> Chemical Science. 6 (1), 50-69 (2015).</li><li>Chen, H., Venkat, S., McGuire, P., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Development+of+Genetic+Code+Expansion+for+Posttranslational+Modification+Studies.">Recent Development of Genetic Code Expansion for Posttranslational Modification Studies.</a> Molecules (Basel, Switzerland). 23 (7), (2018).</li><li>Adumeau, P., Sharma, S. K., Brent, C., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specifically+Labeled+Immunoconjugates+for+Molecular+Imaging--Part+2:+Peptide+Tags+and+Unnatural+Amino+Acids.">Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.</a> Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging. 18 (2), 153-165 (2016).</li><li>Kularatne, S. 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=A+CXCR4-targeted+site-specific+antibody-drug+conjugate.">A CXCR4-targeted site-specific antibody-drug conjugate.</a> Angewandte Chemie (International Ed. in English). 53 (44), 11863-11867 (2014).</li><li>. Patent US20180333484 Polypeptide-Antigen Conjugates with Non-Natural Amino Acids Available from: <a target="_blank" href="https://patentscope.wipo.int/search/en/detail.jsf?docId=US233548973&amp;recNum=65&amp;docAn=15859251&amp;queryString=(GBS)">https://patentscope.wipo.int/search/en/detail.jsf?docId=US233548973&amp;recNum=65&amp;docAn=15859251&amp;queryString=(GBS)</a> (2018)</li><li>Quast, R. B., Mrusek, D., Hoffmeister, C., Sonnabend, A., Kubick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+incorporation+of+non-standard+amino+acids+using+cell-free+protein+synthesis.">Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.</a> FEBS letters. 589 (15), 1703-1712 (2015).</li><li>Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+Genetic+Code+in+Cells+and+Animals:+Biological+Considerations+and+Impacts.">Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts.</a> Accounts of Chemical Research. 50 (11), 2767-2775 (2017).</li><li>Brabham, R., Fascione, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysine+Amber+Stop-Codon+Suppression:+Development+and+Applications.">Pyrrolysine Amber Stop-Codon Suppression: Development and Applications.</a> Chembiochem: A European Journal of Chemical Biology. 18 (20), 1973-1983 (2017).</li><li>. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Genetic+Encoding+of+a+Non-Canonical+Amino+Acid+for+the+Generation+of+Antibody-Drug+Conjugates+Through+a+Fast+Bioorthogonal+Reaction">https://www.ncbi.nlm.nih.gov/pubmed/?term=Genetic+Encoding+of+a+Non-Canonical+Amino+Acid+for+the+Generation+of+Antibody-Drug+Conjugates+Through+a+Fast+Bioorthogonal+Reaction</a> (2019)</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Presolski, S. I., Hong, V. P., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Catalyzed+Azide-Alkyne+Click+Chemistry+for+Bioconjugation.">Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation.</a> Current Protocols in Chemical Biology. 3 (4), 153-162 (2011).</li><li>Prasanna, 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=Semisynthetic+glycoconjugate+based+on+dual+role+protein/PsaA+as+a+pneumococcal+vaccine.">Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine.</a> European Journal of Pharmaceutical Sciences: Official Journal of the European Federation for Pharmaceutical Sciences. 129, 31-41 (2019).</li><li>Morrison, K. 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=Confirmation+of+psaA+in+all+90+serotypes+of+Streptococcus+pneumoniae+by+PCR+and+potential+of+this+assay+for+identification+and+diagnosis.">Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis.</a> Journal of Clinical Microbiology. 38 (1), 434-437 (2000).</li><li>Lin, H., Lin, Z., Meng, C., Huang, J., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+immunogenicity+of+capsular+polysaccharide-surface+adhesin+A+(PsaA)+conjugate+of+Streptococcuspneumoniae.">Preparation and immunogenicity of capsular polysaccharide-surface adhesin A (PsaA) conjugate of Streptococcuspneumoniae.</a> Immunobiology. 215 (7), 545-550 (2010).</li><li>Safari, 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=Identification+of+the+smallest+structure+capable+of+evoking+opsonophagocytic+antibodies+against+Streptococcus+pneumoniae+type+14.">Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14.</a> Infection and Immunity. 76 (10), 4615-4623 (2008).</li><li>Wang, Q., Parrish, A. R., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code+for+biological+studies.">Expanding the genetic code for biological studies.</a> Chemistry &amp; Biology. 16 (3), 323-336 (2009).</li><li>Lawrence, M. C., Pilling, P. A., Epa, V. C., Berry, A. M., Ogunniyi, A. D., Paton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+pneumococcal+surface+antigen+PsaA+reveals+a+metal-binding+site+and+a+novel+structure+for+a+putative+ABC-type+binding+protein.">The crystal structure of pneumococcal surface antigen PsaA reveals a metal-binding site and a novel structure for a putative ABC-type binding protein.</a> Structure (London, England: 1993). 6 (12), 1553-1561 (1998).</li><li>Wright, T. H., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+mutagenesis+for+installation+of+natural+and+unnatural+amino+acid+side+chains+into+recombinant+proteins.">Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins.</a> Nature Protocols. 12 (10), 2243-2250 (2017).</li><li>Dadov&#225;, J., Galan, S. R., Davis, B. G. <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+modified+proteins+via+functionalization+of+dehydroalanine.">Synthesis of modified proteins via functionalization of dehydroalanine.</a> Current Opinion in Chemical Biology. 46, 71-81 (2018).</li><li>Worst, E. 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=Residue-specific+Incorporation+of+Noncanonical+Amino+Acids+into+Model+Proteins+Using+an+Escherichia+coli+Cell-free+Transcription-translation+System.">Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.</a> Journal of Visualized Experiments. (114), (2016).</li><li>Carboni, F., 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=GBS+type+III+oligosaccharides+containing+a+minimal+protective+epitope+can+be+turned+into+effective+vaccines+by+multivalent+presentation.">GBS type III oligosaccharides containing a minimal protective epitope can be turned into effective vaccines by multivalent presentation.</a> The Journal of Infectious Diseases. , (2019).</li></ol>

现场定向诱变是一个直接的策略，将特定的氨基酸纳入一种蛋白质的固定位置，这种蛋白质仍然几乎不用，目的是准备糖基结合疫苗7，8，14。基于20种天然氨基酸方法的经典突变是高效的，因为不需要修改翻译机械。半胱氨酸突变通常针对进一步探索独特的硫醇反应，无论是直接还是分两步（例如，在它修改成去氢碱中...

糖联疫苗是可用于传染病预防治疗的疫苗库的基本要素。在包括幼儿在内的广大年龄组中，它们安全、耐受和高效。它们为脑膜炎球菌、肺炎球菌或乙型流感嗜血杆菌等细胞大 增 菌引起的感染提供了最佳防御。糖糖结合疫苗由纯化细菌多糖制成，形成细菌胶囊或合成寡糖，模仿这些表面表达的多糖2，这些多糖与载体蛋白共价。载体蛋白的存在对于促进针对碳水化合物抗原3所表达的抗原决定因素的保护性体液免疫反应至关重要。除了仔细选择和生产碳水化合物抗原外，已知对糖基结合疫苗的疗效有影响的特征是：载体蛋白的性质、结合化学（包括链接剂的性质和长度（如果使用），或糖/蛋白比3。显然，糖与蛋白质结合的位置以及连接点的数量与免疫原性相关。迄今为止，这两个参数几乎没有被研究，因为糖结合的制备在很大程度上仍然是经验性的。它们的合成通常依赖于使用胺或碳氧酸的功能，分别，赖氨酸或阿斯巴西/谷氨酸侧链残留物存在于载体蛋白序列。这导致的不是单一的，而是糖酸结合的异质混合物。
在蛋白质中氨基酸残留物的活性、可访问性或分布上，产生更明确的糖基结合物，这些糖结合物更可靠，可以记录糖/蛋白质连接4的影响。通过应用蛋白甘油耦合技术，可以朝着这一目标迈出一步，这种重组过程允许在细胞工厂5、6中生产受控糖结合疫苗。然而，糖基化完全发生在D/EXNYS/T sequons内的芦笋残渣（即X是20种天然氨基酸中的任何一种），不是天然存在于载体蛋白上。
站点选择性突变，特别是纳入半胱氨酸，以利用其高度和选择性的活性出现作为替代7，8。生产在序列中加入 UA 的载体蛋白可以为同质糖基结合疫苗制备提供更大的灵活性。超过100个UA已经开发，并进一步纳入各种蛋白质9，10。其中许多含有生物原虫功能，通常用于进行转化后修饰11或移植生物物理探针12或药物13，但非常适合与碳水化合物抗原进一步结合。Biotech14使用无细胞蛋白质合成15，成功的例子已经声称，但根据这一策略制备糖基结合疫苗仍等待推广。
在体内策略用于生产突变载体蛋白需要一种经过改良的转化机制，包括特定的codon、识别codon的tRNA和一种氨基酸-tRNA合成酶（aaRS），它们特别催化了在tRNA上转移的UAA（图1）16。热氨酸琥珀停止抑制是采用UAA的最广泛使用的方法之一，特别是丙丙酰-莱辛（PrK）17。后者反过来可以与azido功能化碳水化合物的哈普顿反应，以提供完全定义的，均匀的糖糖酶。本手稿中，我们描述了如何合成丙丙基-L-lysine，一种携带烷基手柄的UAA，如何在细菌中翻译过程中将其整合到目标蛋白中，最后如何通过点击化学在改性蛋白质和携带亚化物功能的哈顿之间进行结合。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AIM (autoinductif medium)</td>
			<td>Formedium</td>
			<td>AIMLB0210</td>
			<td>Solid powder</td>
		</tr>
		<tr>
			<td>Boc-Lys-OH</td>
			<td>Alfa-Aesar</td>
			<td>H63859</td>
			<td>Solid powder</td>
		</tr>
		<tr>
			<td>BL21(DE3)</td>
			<td>Merck Novagen</td>
			<td>69450</td>
			<td>E. coli str. B, F- ompT gal dcm lon hsdSB(rB-mB-) &#955;(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(&#955;S)</td>
		</tr>
		<tr>
			<td>Dialysis membrane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNAseI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter 0.45 &#181;m</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lysozyme</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>Machery Nagel</td>
			<td>Protino</td>
			<td>Ni-NTA beads in suspension into 20% ethanol</td>
		</tr>
		<tr>
			<td>Pall centrifugal device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET24d-mPsaAK32TAG-ENLYFQ-HHHHHH</td>
			<td>this study</td>
			<td></td>
			<td>same as pET24d-mPsaA-WT but with a K32TAG mutation in the mPsaA gene</td>
		</tr>
		<tr>
			<td>pET24d-mPsaA-WT</td>
			<td>this study</td>
			<td></td>
			<td>pET24d plasmide with the Wt mPsaA gene cloned between the BamHI and XhoI restriction sites with a TEV protease sequence followed by a His6 tag at the C-terminal end of mPsaA gene and carrying the Kanamycine resistance gene</td>
		</tr>
		<tr>
			<td>pEVOL plasmid</td>
			<td>gift fromEdward Lemke EMBL (ref 19)</td>
			<td></td>
			<td>plasmide with p15A origin, two copies of MmPylRS (one under GlnS promoter and one under pAra promoter), one copy of the tRNACUA under the ProK promoter, the chloramphenicol resistance gene</td>
		</tr>
		<tr>
			<td>Propargyl chloroformate</td>
			<td>Sigma-Aldrich</td>
			<td>460923</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Thermo Fisher</td>
			<td>FB120-220</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous glycoconjugate produced by combined unnatural amino acid incorporation and click-chemistry for vaccine purposes

遗传密码扩展是将非自然氨基酸 （UAAs） 引入蛋白质以改变其特性、研究或创建新蛋白质功能或获得蛋白质结合物的有力工具。停止科登抑制，特别是琥珀色的科登抑制，已成为最流行的方法，基因引入UAA在定义的位置。此方法适用于含有生物角功能组的 UAA 的载体蛋白的制备。这种反应性手柄接下来可用于专门和有效地移植合成寡糖，以提供均匀糖结合疫苗。该协议仅限于在1：1碳水化合物哈普顿/载体蛋白比中合成糖基结合物，但可与多对生物激素功能组结合。糖球菌疫苗同质性是确保完整的物理化学特性的重要标准，因此，满足越来越多的要求苛刻的药物监管机构的建议，这是经典结合策略所未达到的标准。此外，该协议使对实际结合疫苗的结构进行微调成为可能，从而产生处理结构-免疫原性关系的工具。

在这个项目中，使用琥珀色停止抑制策略编写了一种均匀糖基结合疫苗，在定义的地点引入 UAA（图1）。肺炎球菌表面阿得辛 A 被选为载体蛋白莫伊蒂。这种蛋白质是高度保存和表达所有菌株肺炎链球菌22。它是高度免疫原性，以前用作载体的肺炎球菌疫苗配方21，23。作为概念的证...

Watch this Scientific Journal Video about Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes at JoVE.com

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

遗传代码扩展用于在定义位点载体蛋白上引入一种非自然氨基酸，该氨基酸具有生物角功能组。生物激素功能进一步用于碳水化合物抗原的位点选择性耦合，以提供均匀糖基结合疫苗。

联合非自然氨基酸合并和点击化学为疫苗目的生产的同质糖共酶

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

该程序的总体目标是通过结合非规范氨基酸和点击化学，生产出同质糖基结合疫苗。遗传密码扩展是将非自然氨基酸整合到蛋白质中以改变其特性、研究或创建新蛋白质功能或获得蛋白质结合物的有力工具。一种在不同位置加入非自然氨基酸的抑制方法已成为最流行的方法。这种方法将在这里应用于一种含有非自然氨基酸的蛋白质载体的产生，该载体含有生物角功能组。这种反应性手柄接下来可用于专门和有效地移植合成寡糖，以提供均匀糖结合疫苗。蛋白碱-莱辛，PrK，从商业Bok-lysine分两步合成。在第一步中，Boc-lysine的未受保护的氨基酸组被转化为丙丙基氯仿酸盐。在第二步中，字母氨基酸组被除保护。合成N（阿尔法）Bo-propargyl-lysine，首先将500毫克的Bok-lysine溶解在5毫升水性一摩尔NaOH和5毫升THF的混合物中，放入烧瓶中，将烧瓶与硅隔膜混合。在冰浴中冷却烧瓶，等待粉末溶解。然后在搅拌下两到三分钟内加入丙基氯仿酸盐滴。在室温下加热反应混合物，继续搅拌10小时。冷却二乙醚、水性一摩尔盐酸和乙酸乙酯的溶液。冷却冰浴中的粗反应混合物。将混合物倒入分离漏斗中。提取五十毫升二乙醚的混合物。萃取可能导致压力积聚，请确保经常释放任何压力积聚。收集水相并丢弃有机层。在分离漏斗中的水相中小心地加入五十毫升一摩尔盐酸。然后，使用乙酸乙酯的三十毫升提取水层两次。收集有机相，通过 TLC 验证您的化合物是否存在。在这个阶段，它应该处于有机阶段。将组合的有机层干燥在硫酸镁上。过滤掉固相。在旋转蒸发器上在压力下将滤液集中。溶解脱盐氯仿中原油N（阿尔法）Bo-Propargyl-lysine的样品，通过NPR控制其特性。要合成丙丙基-L-莱辛，在装有隔膜的圆形纽扣瓶中引入N（阿尔法）Boc-propargyl-lysine。在阿贡下的烧瓶中加入四毫升无水二氯甲烷。在搅拌下加入滴，四毫升三氟乙酸。更换玻璃盖的硅隔膜，以避免 TFA 损坏硅隔膜。在室温下搅拌反应混合物一小时。验证 TLC 对 Boc-PrK 的保护。在减压下浓缩反应混合物。在粗残留物中加入二乙醚。在褶皱玻璃上以白色固体的形式过滤蛋白丙基-L-莱辛。溶解氧化铝二甲酸二甲酸的一等素，然后进行 NMR 分析，以控制其特性和纯度。然后，丙丙丙基-L-莱辛在蒸馏水中溶解，最终浓度为100毫升，以零下20度储存在一毫升等分中。要将质粒共同转化为表达菌株，在五分钟内在冰上解冻一百微升的化学能力E.Coli BL21。将每个质粒的一微升或每粒质粒的50至100纳米添加到细胞中，并在冰上孵育30分钟。在45秒内在42度孵育称职细胞。然后，把它们放回冰上两分钟。加入900微升LB介质，在37度的震动下孵育一小时，以允许抗生素表达。然后用抗生素将转化后的细菌放在LB加糖上。让细菌在37度下一夜之间生长。为了表达用PrK修饰的蛋白质，用抗生素在5毫升LB介质中接种单个共转化菌落。在37度下孵育过夜，摇晃。第二天，用5毫升原培养基稀释含有抗生素的500毫升、0.02%的L-arabinose和1毫升的PrK，并在37度下孵育24小时。通过执行没有PrK的培养物，包括负对比，通过执行含有野生型蛋白质基因的克隆的培养，包括负对比。在10分钟内以5000xg的离心从隔夜培养中收获细胞。丢弃上一液，将颗粒冻结在零下20度。为了通过重力流板凳亲和力色谱纯化蛋白质，将细胞颗粒重新排入二十毫升的解解缓冲液中。加入DNase I和解酶，在30分钟内将悬浮液孵育为37度，允许解解。在五分钟内将细胞在冰中声波化，然后在30分钟内以20，000xg的离心去除细胞碎片。用0.45微米过滤器过滤酸盐，将Ni-NTA树脂加入悬浮液。在四度下轻轻混合一小时。将悬浮液倒入聚丙烯柱中，并收集未绑定分数。用十毫升洗涤树脂，然后用五毫升的洗涤缓冲液清洗。收集洗涤分数。用一毫米洗脱缓冲液来洗取他标记的蛋白质，重复这一步四次，并收集所有额外的分数。将含有纯组蛋白标记蛋白质的馏分混合，使用透析膜将其透析到一升TEV蛋白酶缓冲液中过夜。将蛋白质样本收集到50毫升管中，并加入TEV缓冲液，在1毫升的最终体积中获得每毫升2毫克的最终浓度。添加一百微升的 TEV 蛋白酶。添加 50 微升 0.1 摩尔 DTT。配有高达五毫升的 TEV 缓冲液。在四度孵育过夜，缓慢摇晃。为了去除EDTA，使用透析膜和磷酸盐缓冲液，使用5毫摩尔伊米达佐尔，在一夜之间将蛋白质解入四度。为了消除TEV蛋白酶和未消化的蛋白质，与Ni-NTA珠子孵育混合物，并在四度下轻轻混合一小时。将悬浮液倒入聚丙烯柱中。收集了未绑定分数。TEV蛋白酶和未消化的蛋白质与珠子保持结合，消化的蛋白质被洗出。用五毫升的洗涤缓冲液清洗柱子，并收集洗涤分数。透析消化的蛋白质对一升点击缓冲液在四度过夜与透析膜，以去除伊米达佐，以及交换缓冲液。测量蛋白质在280纳米的浓度。为了将mPsaA与6-六氯氟辛-阿齐德或解毒剂功能化碳水化合物抗原结合，将浓度为57.8微摩尔的PrK突变蛋白放入两毫升微管中。加入10微升5毫摩尔阿齐德化合物，然后加入硫酸铜溶液和THPTA的预混合。添加盐酸氨基瓜尼丁。加入临时准备的抗酸钠水溶液。关闭管子，通过反转几次混合，并在室温下孵育两个小时。通过添加 EDTA 停止反应。检查 SDS 页面上的结合。迁移后，将凝胶在 312 纳米的紫外光上可视化，以表示与荧光素的结合。或用库马西蓝染色凝胶，以可视化与碳水化合物抗原的结合。作为哈彭的添加应诱导分子量的变化。通过将糖结合应用于与PBS平衡的凝胶过滤柱中，净化糖基结合。收集含有糖结合的馏分。对于长期储存，将糖分联对着蒸馏水进行透析，然后冷冻干燥，将糖结合物储存在零下80度。PrK 在位置 32 被引入， 以取代 Psaa N 术语附近的莱辛。通过SDS页面和西方印迹分析，使用抗西丁标签抗体检查mPsaA生产的疗效。全长蛋白质的存在强烈地表明PrK的成功整合。然而，强度低于野生型mPsa观察到的强度。mPsa（K32PrK）在Ni-NTA珠上被净化。以8毫克的典型产量和PrK残留物的合并最终通过质谱法确认。使用 Tev 蛋白酶在蛋白酶的蛋白酶裂解时去除的 Histidine 标签。使用阿齐多功能化荧光素评估碱基（K32PrK）的活性，进一步用于结合合成寡糖抗原。实验与野生型mPsa相比是一种控制。最后，糖联聚酯通过凝胶过滤进行纯化，其特性通过质谱法得到确认。在这个项目中，使用琥珀色停止胶质抑制技术准备了均质糖基结合疫苗，在定义的位点下加入一种非自然氨基酸。糖联琼疫苗同质性是保证物理化学特性完整的重要标准。因此，满足越来越多的要求苛刻的药检机构的建议。使用经典的纯结合法不满足此标准。此外，该协议使得可以微调糖结合疫苗的结构。产生一个前所未有的工具，研究同质性与糖酸结合结构之间的关系。

Research

JoVE Journal

Bioengineering

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

科研

生物工程

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...