Name: expression, purification, and antimicrobial activity of s100a12
Uploaded: 2017-05-13T13:00:00.000+00:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Expression, Purification, and Antimicrobial Activity of S100A12 at JoVE.com

Esta pesquisa foi apoiada pelo Departamento de Veteranos Assuntos Carreira Desenvolvimento Award 1IK2BX001701, CTSA prêmio UL1TR000445 do Centro Nacional para o Avanço Translational Sciences, o National Science Foundation Award Números 1547757 e 1400969, e NIH concessão GM05551. Seu conteúdo é exclusivamente da responsabilidade dos autores e não representam necessariamente visões oficiais do Centro Nacional para o Avanço das Ciências Translacionais ou dos Institutos Nacionais de Saúde.

1. Expressão de S100A12 <ol><li> Transformar células BL21 DE3 competentes com um plasmídeo pGEMEX-S100a12 utilizando um protocolo padrão de choque térmico 18 . Adicionar 1 a 5 μL de plasmídeo a 50 μL de bactérias num tubo de microcentrífuga em gelo. Incubar durante 20 min. </li><li> Aquecer as células a 42 ° C durante 30 s. </li><li> Incubar as células em gelo durante 2 min. </li><li> Adicionar 500 μL de meio SOC às células. Incubar a 37 ° C com agitação a 250 rpm num agitador orbital durante 1 h. </li><li> Placa 150 μL da reação de transformação em meio LB-agar (suplementado com 100 μg / mL de ampicilina). Incubar durante 12-16 h a 37 ° C. </li><li> Escolha uma colônia. Inocular 2 mL de LB (suplementado com 100 μg / mL de ampicilina). </li><li> Incubar durante 4-6 h a 37 ° C num agitador orbital (300 rpm). A OD 600 deve ler entre 1-3 unidades de absorvância. </li><li> Adicionar 500 μL de cultura inicial a 50 mL de ZYM-5052 autoinduCtion 26 suplementado com 100 μg / mL de ampicilina. Para melhor arejamento e máxima expressão, use um frasco Erlenmeyer de 250 mL. Agitar (300 rpm) durante 24 h, a 37 ° C. </li><li> Transferir a suspensão bacteriana para um tubo de centrífuga. Pellet as células por centrifugação (4.000 xg, 10 min) a 4 ° C. </li><li> Decantar a mídia, registrar a amostra e armazenar a pasta celular a -80 ° C. A amostra é estável por anos. </li></ol> 2. Purificação de S100A12 utilizando cromatografia de baixa pressão <ol><li> Ressuspender as células em 30 mL de Tris 20 mM, pH 8,0. </li><li> Sonicate a suspensão em gelo para lisar células. Utilize ~ 20 W de saída, 5 s ligado e 5 s fora do ciclo durante 5 min. </li><li> Transferir a solução para tubos de centrífuga de alta velocidade. Esclarecer o lisado celular por centrifugação, 20.000 xg durante 30 min a 4 ° C. </li><li> Decantar o sobrenadante e transferir para um copo de polipropileno limpo de 100 mL. Arrefecer a solução colocando o copo em gelo. Adicionar uma agitaçãoR e adicionar lentamente 11,20 g de sulfato de amónio. Deixar a solução agitar sobre gelo durante mais 1 h. Isto irá criar uma solução a 60% de sulfato de amónio e precipitar a maioria das proteínas endógenas de E. coli . S100A12 permanecerá solúvel. </li><li> Centrifugar a solução a 4 ° C, 20 000 xg durante 20 minutos para sedimentar a proteína precipitada. </li><li> Decantar o sobrenadante e transferir para tubos de diálise (MWCO 3.500 kDa). Dializar contra 1 L de Tris 20 mM, pH 8,0 a 4 ° C. Mude o tampão de diálise duas vezes. Permitir 4 h entre as alterações. </li><li> Cromatografia de permuta aniónica <ol><li> Realizar a cromatografia em um sistema de baixa pressão. O caudal típico é de 1 mL / min. </li><li> Equilibrar uma coluna de 5 mL de Sepharose com 10 mL de Tris 20 mM, pH 8,0. </li><li> Coloque os ~ 40 mL de solução de S100A12 usando a bomba de amostra (colete o fluxo através). </li><li> Lavar a coluna com 10 mL de Tris 20 mM, pH 8. </li><li> Desenvolver a coluna com um gradiente 0-30% (BuFfer B é Tris 20 mM, pH 8,0, NaCl 1 M) ao longo de 19 volumes de coluna (CV, 95 mL). Recolher fracções de 5 mL. </li><li> Tomar uma alíquota de 10 μL de cada fracção e analisar utilizando MES SDS PAGE com coloração Coomassie. Executar o gel utilizando uma tensão constante (20 V / cm) durante 30 min. </li><li> Pool fracções contendo S100A12. S100A12 corre a ~ 10 kDa de proteína num gel desnaturante. </li><li> Concentrar as fracções para 5 mL utilizando um dispositivo de ultrafiltração (MWCO 10 kDa). Centrifugar a 3000 xg durante ~ 8 min. Pegue a fração superior. </li></ol></li><li> Cromatografia de exclusão de tamanho <ol><li> Equilibrar a coluna S75 com 1 CV (120 mL) de Tris 20 mM pH 8, NaCl 100 mM. </li><li> Injectar &lt;5 mL de fracções S100A12 concentradas (a partir da cromatografia de permuta iónica). </li><li> Desenvolver a coluna a um caudal de 1 mL / m em 120 mL. Recolher fracções de 5 mL. </li><li> Tomar uma alíquota de 10 μL de cada fracção e analisar utilizando SDS PAGE com coloração com Coomassie. Validar a identidade proteica usandoWestern blot ou espectrometria de massa (massa molecular calculada da subunidade monomérica 10,575.0 Da, medida 10575,4 Da). </li></ol></li><li> Frações de pool <ol><li> Medir a absorvï¿½cia da proteï¿½a A 280 utilizando um espectrofotï¿½etro. Use o buffer SEC como um espaço em branco. O coeficiente de extinção calculado para o homodímero S100A12 é 5960 M -1 cm -1 . NOTA: Rendimentos típicos são 35-45 mg de S100A12 por 50 mL de cultura. </li><li> Alíquota S100A12 em tubos de microcentrífuga de 1,5 mL (1 mg / tubo), congelamento rápido em azoto líquido e armazenar a -80 ° C. </li></ol></li></ol> 3. Ensaios de actividade antimicrobiana <ol><li> Colocar a estirpe G27 de H. pylori sobre placas de ágar de soja trípticas suplementadas com sangue de ovelha a 5% (placas de ágar sangue). Crescer 2-3 dias a 37 ° C em ar ambiente suplementado com 5% de dióxido de carbono. </li><li> Inocular H. pylori em caldo de Brucella suplementado com 1x colesterol. Cultura sobreAgitação noturna (250 rpm) a 37 ° C em ar ambiente suplementado com 5% de dióxido de carbono. </li><li> Diluir H. pylori 1:10 em 50% de caldo de Brucella, 50% de tampão de calprotectina mais 1x colesterol e cultura em meio isolado ou suplementado com 100 μM de cloreto de zinco mais 0, 100 ou 1000 μg / mL de S100A12 purificado. Cultura agitação durante a noite (250 rpm) a 37 ° C em ar ambiente suplementado com 5% de dióxido de carbono. </li><li> No dia seguinte, efectuar diluições em série e placa sobre placas de agar de sangue. Deixar as colónias bacterianas crescerem durante 2-3 dias a 37 ° C em ar ambiente suplementado com 5% de dióxido de carbono. Enumerar as unidades formadoras de colónias para calcular o crescimento bacteriano na presença ou ausência de S100A12 e / ou zinco exógeno. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

É apresentado um protocolo eficiente para expressão e purificação de S100A12 humano. O sistema de expressão de E. coli é a ferramenta mais comum usada para a produção de proteínas recombinantes, particularmente quando são necessárias quantidades de mg para estudos bioquímicos e biofísicos. Um aperfeiçoamento chave do procedimento aqui descrito é o uso de meios de auto-indução 26 que aumenta o rendimento da proteína purificada por um factor de quase trinta em comparaçã...

As proteï¿½as S100 sï¿½ uma classe da famï¿½ia EF-hand de proteï¿½as de ligaï¿½o de cï¿½cio com uma diversidade de funï¿½es 1 . Eles são expressos de uma forma específica de tecidos e células, e regulam um amplo espectro de funções celulares 2 , 3 . Exclusivas para proteínas de ligação ao cálcio, as proteínas S100 exibem funções intracelulares e extracelulares 4 , 5 . Dentro da célula, a ligação de Ca2 + induz uma alteração conformacional que expõe uma superfície hidrofóbica que alveja especificamente parceiros de ligação à proteína 6 . Este mecanismo intracelular regula processos importantes como proliferação celular, diferenciação e metabolismo energético. No meio extracelular, as proteínas S100 exibem duas funções 7 . Num deles, actuam como proteínas associadas a padrões moleculares associados (DAMP) e iniciam uma pró-inflamaçãoResposta imune atórica através da interação com receptores de reconhecimento de padrão 8 , 9 . Além disso, vários membros da classe de proteínas S100 sequestam metais de transição, uma função que serve para privar patógenos microbianos em um processo denominado imunidade nutricional 10 , 11 . S100A12 (também conhecida como calgranulina C e EN-RAGE) é altamente expressa em macrófagos e neutrófilos e tem sido identificada como potencial biomarcador para doenças inflamatórias 12,13 . Além de ligar o cálcio nos seus locais EF-Hand, S100A12 tem dois locais de ligação de metais de transição de alta afinidade localizados em extremidades opostas da interface dimérica 14 , 15 . Cada sítio de ligação é constituído por três resíduos de histidina e um resíduo de ácido aspártico e pode quelar zinco ou cobre &lt;Sup class = &quot;xref&quot;&gt; 16 , 17 . Recentemente, relatamos que a fome de zinco dependente de S100A12 é importante na regulação do crescimento de Helicobacter pylori e da atividade de fatores de virulência pró-inflamatórios 18 . H. pylori infecta o estômago de cerca de metade da população humana do mundo; Tornando-se sem dúvida um dos mais bem sucedidos patógenos bacterianos [ 19] . A infecção com H. pylori pode levar a resultados significativos da doença gástrica incluindo gastrite, úlcera péptica e duodenal, linfoma de tecido linfóide associado à mucosa (MALT) e adenocarcinoma gástrico invasivo (câncer de estômago). O câncer de estômago é a principal causa de morte associada ao câncer de não-cardia no mundo, eo maior fator de risco associado para câncer de estômago é a infecção por H. pylori . H. pylori persiste no nicho gástrico apesar de um robuSt resposta imune ao patógeno, sublinhando a necessidade de uma melhor compreensão dos mecanismos imunes de controle desta infecção bacteriana 20 , 21 , 22 , 23 . A inflamação associada ao H. pylori é caracterizada por uma profunda infiltração de células polimorfonucleares, ou neutrófilos, que depositam um repertório de proteínas antimicrobianas, incluindo S100A12, no local da infecção 18 , 24 , 25 . Em um esforço para entender o complexo diálogo entre hospedeiro e patógeno, buscamos refinar a técnica para purificar S100A12 e usá-lo para estudar o efeito antimicrobiano que exerce sobre este patógeno clinicamente relevante. O protocolo abaixo descreve uma técnica melhorada para a purificação de S100A12 no seu estado biologicamente activo; Capazes de se ligar a metais nutrientes com altaE eliminando-os de microrganismos invasores. Além disso, os métodos abaixo destacam a utilidade desta proteína como um reagente crítico para estudar o mecanismo pelo qual as moléculas antimicrobianas inatas restringem o crescimento de patógenos bacterianos. As proteínas da família S100A ganharam apreciação como um importante grupo de moléculas do sistema imune inatas que participam na sinalização imune bem como na defesa do hospedeiro 27 . O mais bem estudado destes é a calprotectina (MRP-8/14, calgranulina A / B, S100A8 / A9) 28 , 29 , 30 . A calprotectina é uma proteína associada a neutrófilos que forma um heterodímero das subunidades S100A8 e S100A9 que liga metais de transição na interface dímero 31 . Verificou-se que a calprotectina possui dois locais de ligação de metal: o local 1 pode ligar Zn2 + , Mn2 + ou Fe2 + e o sítio 2 pode<html> Se Zn2 + 31 , 32 . Numerosos relatos demonstraram que a calprotectina tem atividades antimicrobianas contra diversos agentes patogênicos incluindo Staphylococcus aureus , Candida albicans , Acinetobacter baumannii , Klebsiella pneumoniae , Escherichia coli e H. pylori e que os efeitos inibitórios são devidos à atividade de quelação de metal da calprotectina 25,28 , 29 . Trabalhos anteriores demonstraram que a calprotectina exerce numerosas atividades sobre H. pylori, incluindo a alteração da estrutura do lípido A na membrana externa, reprimindo o sistema de secreção tipo IV (que é um importante fator de virulência proinflamatório dentro de H. pylori ), induzindo a formação de biofilme e reprimindo Crescimento e viabilidade de H. pylori de uma forma dependente da doseClass = &quot;xref&quot;&gt; 25 , 33 . Além disso, os ensaios genéticos e bioquímicos revelaram que a actividade antibacteriana da calprotectina contra H. pylori foi largamente derivada da sua capacidade para ligar o zinco nutriente 25 . H. pylori requer zinco, como foi determinado por pesquisas anteriores, que utilizou um meio quimicamente definido para determinar os requisitos de micronutrientes para este patógeno para crescer e proliferar [ 34] . Além disso, a calprotectina foi altamente abundante nos tecidos infectados com H. pylori e associada a infiltrados neutrófilos, indicando que o hospedeiro pode empregar calprotectina como estratégia antimicrobiana durante a infecção e subseqüente inflamação 25,35. Evidências recentes de técnicas de triagem proteômica não-direcionadas sugerem que sob condições onde espécies reativas de oxigênio são abundantes, calprotecO estanho sofre modificações pós-translacionais que alteram o local de ligação da hexa-histidina, inibindo assim a actividade de ligação do metal da proteína 36 . Como tal, nós hipotetizamos que outras proteínas da família S100A entre o vasto repertório dessas moléculas poderiam potencialmente atuar como quelantes metálicos auxiliares. Seleccionou-se S100A12 para estudo adicional porque não foi identificado no referido ecrã para modificação pós-tradução, tem a capacidade de ligar o zinco e é altamente abundante em tecidos humanos derivados de indivíduos infectados com H. pylori . O nosso trabalho indica que S100A12 pode inibir o crescimento ea viabilidade de H. pylori de uma forma dependente da dose na estirpe G27 de H. pylori , e que a actividade antimicrobiana desta proteína pode ser invertida pela adição de zinco em excesso de nutrientes. Este trabalho complementa o nosso trabalho anterior indicando S100A12 exerceram atividade antimicrobiana contra PMSS1 e 7.13 de H. pylori , demonstrando sua ampla atividade antibacteriana contra numerosos isolados clínicos e linhagens de H. pylori adaptadas ao laboratório 18 . Juntos, estes resultados confirmam a importância de S100A12 como um mecanismo para controlar o crescimento bacteriano e proliferação via imunidade nutricional. Futuros estudos desta importante interacção hospedeiro-bactéria poderiam incluir explorar a actividade de S100A12 para reduzir a carga bacteriana dentro dos tecidos do hospedeiro ou determinar a contribuição desta proteína para a sinalização imune no contexto da infecção por H. pylori .

<table><tbody><tr><td>&lt;strong&gt;S100A12 Expression&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Expression cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BL 21 (DE3) competent cells</td><td>New England Biolabs</td><td>C25271</td><td></td></tr><tr><td>Autoinduction medium components</td><td></td><td></td><td></td></tr><tr><td>Yeast Extract</td><td>Research Products International</td><td>Y20020-250.0</td><td></td></tr><tr><td>NaCl</td><td>Research Products International</td><td>S23020-500.0</td><td></td></tr><tr><td>Tryptone</td><td>Research Products International</td><td>T60060-250.0</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>F2877</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>M65240-100.0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>S23100-500.0</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>P41200-500.0</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Research Products International</td><td>A20424-500.0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>S25150-500.0</td><td></td></tr><tr><td>Glycerol</td><td>Research Products International</td><td>G22020-1.0</td><td></td></tr><tr><td>D-glucose</td><td>Research Products International</td><td>G32040-500.0</td><td></td></tr><tr><td>lactose</td><td>Sigma-Aldrich</td><td>L2643</td><td></td></tr><tr><td>&lt;strong&gt;Selection agent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ampicillin</td><td>Research Products International</td><td>A40040-5.0</td><td></td></tr><tr><td>&lt;strong&gt;S100A12 Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AKTA Start Chromatography System</td><td>GE</td><td>29-220-94</td><td></td></tr><tr><td>HiTrap Q Sepharaose Fast Flow</td><td>GE</td><td>17-5156-01</td><td></td></tr><tr><td>HiPrep 16/60 S-200 HR</td><td>GE</td><td>17-1166-01</td><td></td></tr><tr><td>Nanodrop lite spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-LITE</td><td></td></tr><tr><td>Tris Base</td><td>Research Products International</td><td>T60040-1000.0</td><td></td></tr><tr><td>&lt;em&gt;H. pylori &lt;/em&gt;Culture</td><td></td><td></td><td></td></tr><tr><td>Blood agar plates</td><td>Lab Supply Company</td><td>BBL221261</td><td></td></tr><tr><td>Brucella broth</td><td>Sigma-Aldrich</td><td>B3051</td><td></td></tr><tr><td>Cholesterol (250x)</td><td>Thermo Fisher Scientific</td><td>12531018</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>793566</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Zinc Chloride</td><td>Sigma-Aldrich</td><td>229997</td><td></td></tr></tbody></table>

expression, purification, and antimicrobial activity of s100a12

As proteínas de calgranulina são mediadores importantes da imunidade inata e são membros da classe S100 da família EF-mão de proteínas de ligação de cálcio. Algumas proteínas S100 têm a capacidade de se ligar a metais de transição com alta afinidade e efetivamente seqüestrar eles longe de patógenos microbianos invasores em um processo que é denominado &quot;imunidade nutricional&quot;. O S100A12 (EN-RAGE) liga tanto o zinco como o cobre e é altamente abundante em células imunes inatas tais como macrófagos e neutrófilos. Relatamos um método refinado para a expressão, enriquecimento e purificação de S100A12 na sua configuração ativa de ligação a metais. A utilização desta proteína em análises de crescimento e viabilidade bacteriana revela que S100A12 tem actividade antimicrobiana contra o patogénio bacteriano Helicobacter pylori . A actividade antimicrobiana baseia-se na actividade de ligação ao zinco de S100A12, que quela o zinco nutriente, privando assim H. pylori que requer zinco para crescimento e pRoliferation.

S100A12 expressão e purificação Uma purificação em três passos produziu ~ 40 mg de S100A12 recombinante a partir de 50 mL de cultura bacteriana. O primeiro passo foi uma precipitação com sulfato de amónio de proteínas endógenas de E. coli . Este passo foi seguido por cromatografia de permuta aniónica ( Figura 1A ). A proteína é rastreada por uma SDS-PAGE corada com Coomassi...

Watch this Scientific Journal Video about Expression, Purification, and Antimicrobial Activity of S100A12 at JoVE.com

Expression, Purification, and Antimicrobial Activity of S100A12

Aqui, apresentamos um método para expressar e purificar S100A12 (calgranulina C). Descrevemos um protocolo para medir sua atividade antimicrobiana contra o patógeno humano H. pylori .

Expressão, Purificação e Actividade Antimicrobiana de S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

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8.3K visualizações.  Fisk University.  Aqui, apresentamos um método para expressar e purificar S100A12 (calgranulina C). Descrevemos um protocolo para medir sua atividade antimicrobiana contra o patógeno humano H. pylori . 

Vídeo: Expression, Purification, and Antimicrobial Activity of S100A12

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Expressão bacteriana e purificação da matriz humana Metaloproteinase-3 usando cromatografia de afinidade

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Bioquímica

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

Um pipeline para investigar as estruturas e caminhos de sinalização de receptores sphingosine 1-fosfato

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Implementação de um Sistema de Infecção baseado em Inserir Permeável Membrana para estudar os efeitos de secretada bacterianas toxinas em células hospedeiras de mamíferos

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Imunologia e Infecção

Purificação de uma enzima fibrinolítica de Sipunculus nudus via cromatografia de afinidade

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Autor em destaque: Purificação de afinidade de uma enzima fibrinolítica de Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

Purificação por Afinidade de uma Enzima Fibrinolítica de Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

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

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Peptídeos antimicrobianos produzidos pela incorporação de pressão seletiva de aminoácidos não-canônicos

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

Bioengenharia

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.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

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

Biologia

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

This protocol describes a purification method for recombinant tag-free calcium binding protein S100A12 and its ion-induced oligomers for human monocyte stimulation assays.

Purificação do S100A12 humano e seus Oligomers íon-induzidos para a estimulação imune da pilha

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Expressão, Purificação e Actividade Antimicrobiana de S100A12

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Purificação do S100A12 humano e seus Oligomers íon-induzidos para a estimulação imune da pilha

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais