BRPI0611376A2 - processo para a producão de proteìna c ativada humana para o tratamento de sepsia - Google Patents
processo para a producão de proteìna c ativada humana para o tratamento de sepsia Download PDFInfo
- Publication number
- BRPI0611376A2 BRPI0611376A2 BRPI0611376-1A BRPI0611376A BRPI0611376A2 BR PI0611376 A2 BRPI0611376 A2 BR PI0611376A2 BR PI0611376 A BRPI0611376 A BR PI0611376A BR PI0611376 A2 BRPI0611376 A2 BR PI0611376A2
- Authority
- BR
- Brazil
- Prior art keywords
- leu
- glu
- gly
- arg
- asp
- Prior art date
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Abstract
A presente invencao refere-se a um método recombinante de producao de proteína C ativada. A invencao refere-se a um método de construcao, transformacóo, expressao, purificacao e producao de proteína C humana ativada recombinante. Construcões de DNA compreendendo os elementos de controle associados ao gene de interesse foram descritas. A seqÕencia de ácido nucléico de interesse teve o codon otimizado para permitir expressao nas células hospedeiras de mamífero adequadas.
Description
Relatório Descritivo da Patente de Invenção para "PROCESSOPARA A PRODUÇÃO DE PROTEÍNA C ATIVADA HUMANA PARA OTRATAMENTO DE SEPSIA".
Campo da Invenção
A presente invenção refere-se a um método recombinante deprodução de Proteína C ativada. A invenção refere-se a um método de cons-trução, transformação, expressão, purificação e produção de proteína C hu-mana ativada recombinante. Construções de DNA compreendendo os ele-mentos de controla associados ao gene de interesse foram descritas.
A seqüência de ácido nucléico de interesse teve o códon otimizado parapermitir expressão em células hospedeiras de mamífero adequadas.Antecedentes da Invenção
Xigris (Drotrecogina alfa) é uma forma recombinante de ProteínaC Ativada humana. Ela é uma protease de serina com a mesma seqüênciade aminoácido que a Proteína C Ativada derivada de plasma humano. Prote-ína C Ativada é um modulador importante da resposta sistêmica à infecção etem propriedades antitrombóticas, pró-fibrinolíticas e antiinflamatórias.
A Drotrecogina alfa (ativada) é uma glicoproteína com peso molecular deaproximadamente 55 kD. A forma precursora de Proteína C contém um pep-tídeo líder pre pro (ausente na proteína madura), um domínio de ácido γ-carboxiglutâmico (GIa) de 9 resíduos de Gla, uma pilha de aminoácido hidro-fóbico helicoidal curto, dois domínios semelhantes ao fator de crescimentoepidérmico (EGF), um peptídeo de ligação entre as cadeia leve e pesada,um peptídeo de ativação e um domínio semelhante à tripsina SP no qual atríade catalítica está localizada em His-211, Asp-257 e Ser-360. A principalfunção do domínio de EGF é proporcionar interações proteína-proteína ouproteína-célula. Também foi mostrado que os resíduos presentes no motivoEGF interagem funcionalmente com diferentes ativadores e substratos. Alémdisso, a hélice de conexão tem resíduos que participam na coordenação deíons de cálcio ligados ao domínio EGF-I que é considerado como exercendoum papel neuroprotetor.
Modificações pós-traducionais remover o dipeptídeo Lys-156-Arg-157, de modo que a forma com uma única cadeia é convertida em umamolécula com duas cadeias ligadas por uma ligação de dissulfeto. 80% dozimógeno PC estão nessa forma. Também, carboxilação de resíduos de áci-do glutâmico no domínio Gla amino terminal, hidroxilação de um resíduo Aspno domínio EGF-I e glicosilação são outros eventos pós-traducionais.RhAPC e APC derivada de plasma humano têm os mesmos sítios de glicosi-lação, embora algumas variações existam nas estruturas de glicosilação.
APC humana tem quatro sítios de N-glicosilação ligados à asparagina. Elatem um teor de ácido siálico cinco vezes maior comparado com outras prote-ínas do plasma.
APC humana tem quatro sítios de N-glicosilação ligados à aspa-ragina. Ela tem um teor cinco vezes maior de fucose e um teor duas vezesmaior de ácido siálico comparado com outras proteínas do plasma. ProteínaC Ativada atua através de inibição de Fatores Va e VlIIa. Dados in vitro indi-cam que a Proteína C Ativada tem atividade pró-fibrinolítica indireta atravésde sua capacidade de inibir o inibidor de ativador de plasminogênio 1 (PAI-1)e limitar a geração de inibidor de fibrinólise trombina-ativável ativado. Adicio-nalmente, dados in vitro indicam que a Proteína C Ativada pode exercer umefeito antiinflamatório através de inibição da produção de fator de necrose detumor humano por monócitos, através de bloqueio da adesão de leucócitos àselectinas e através de limitação das respostas inflamatórias trombina-induzidas dentro do endotélio microvascular.
Vários métodos foram descritos para a expressão de proteínasrecombinantes em sistemas eucariotas superiores. Sistemas de expressão decélulas CHO-K1, HEK293 (e variantes) foram agora estabelecidos como ossistemas predominantes de escolha para expressão de proteína de mamífero.
O procedimento esboçado é adequado para a transfecção de seqüência deácido nucléico sintetizada de novo que codifica a Drotrecogina alfa humanarecombinante em hospedeiros mamíferos adequados para expressão.
O procedimento esboçado abaixo é adequado para a produçãode proteína C humana ativada recombinante solúvel bioativa. Os protocolosatuais fazem uso de uma linhagem de células humanas estabelecida possu-indo o DNA complementar para o zimógeno de proteína C humana inativoque secreta a proteína no meio de fermentação. Proteína C humana é enzi-maticamente ativada através de clivagem com alfa-trombina, tripsina, ativa-dor de fator X de veneno de víbora de Russell ou uma mistura de trombina etrombomodulina para obter proteína C ativada e, subseqüentemente, purifi-cada. Contudo, esses procedimentos de ativação envolvem o risco de con-taminação e maiores custos de produção. A presente investigação objetiva aprodução da proteína C ativada diretamente a partir das células recombinan-tes através da incorporação da protease célula-associada.
Tais proteases poderiam estar localizadas no citoplasma ou or-ganela celular ou nas membranas celulares que podem clivar proteínas du-rante ou imediatamente quando de secreção. Conseqüentemente, a estraté-gia tem sido empregada para a produção de proteína C ativada recombinan-te diretamente quando de secreção de uma célula hospedeira eucariota, istoé, HEK293.
A enzima recombinante será indicada para uso na redução demortalidade em pacientes adultos com sepsia grave (isto é, sepsia associa-da à insuficiência aguda de órgãos) que têm um alto risco de morte.
Descrição das Figuras Incluídas
Figura 1 - Alinhamento de seqüência aos pares das versões comcódon não-otimizado e otimizado da seqüência de nucleotídeo de DNA quecodifica Drotrecogina alfa ou Xigris.
Figura 2 - Fragmentos digeridos por restrição purificados em gelde cDNA de DROT e pcDNA3.1 D/V5-His.
Figura 3 - Análise através de digestão por restrição de clonesputativos de AVCIPpcDNA3.1 DA/5-His/Xigris.
Figura 4 - Análise de digestão por restrição de clones AV-CIPpcDNAS.1D/VS-His/Xigris usando enzimas que clivam cDNA de pcD-NA3.1-DROT internamente.
Figura 5 - Alinhamento de seqüência do pcDNA3.1-DROT (Xi-gris sintético) sintetizado de novo com a seqüência estabelecida do gene deXigris.Figura 6 - Alinhamento de seqüência do pcDNA3.1-DROT-Opt(Xigris-Opt sintético) sintetizado de novo com a seqüência estabelecida dogene de Xigris-Opt.
Figura 7 - Alinhamento de seqüência do clone de cDNA de pcD-NA3.1DROT-/V5-His/Xigris Ng 4 com a seqüência estabelecida do gene deXigris.
Figura 8 - Mapa de Estrutura: pcDNA3.1-DROT-D/V5-His/Xigris.Listagem de Seqüência
SEQ ID N9 1: seqüência de nucleotídeo da Proteína C Ativada
SEQ ID N9 2: seqüência com códon otimizado da Proteína C Ativada
Sumário da Invenção
Construções de DNA compreendendo os elementos de controleassociados ao gene de interesse as quais permitem expressão do gene deinteresse foram descritas. Ainda outro aspecto da invenção é a otimizaçãode códon do ácido nucléico sintetizado de novo para permitir expressão domesmo em células de mamífero. A seqüência com códon otimizado é trans-formada em linhagens de células de mamífero adequadas para expressão.
Descrição Detalhada da Invenção
Exemplo 1:
O design do vetor de expressão de mamífero para a expressãode proteína C humana recombinante (ativada) foi modificado para acomodarquatro sítios de N-glicosilação N-Iigados e são baseados em um dos vetorescomercialmente disponíveis (EX: pcDNA ou plfíES da Invitrogen ou BD Bi-osciences, respectivamente), modificados para incluir as seguintes caracte-rísticas:
(a) um sítio de clonagem múltipla para inserção do cDNA de pro-teína C humana, incluindo seu peptídeo sinalizador natural.
(b) O design do vetor de expressão também acomoda uma co-expressão IRES-mediada independente (bicistrônica) da proteína verde fluo-rescente, a qual permitiria seleção rápida de transfectantes com alta expres-são usando seleção de células fluorescência-assistida.Síntese da Estrutura de Fusão
Abordagem de novo
Uma abordagem de novo em termos de síntese das regiões decodificação da estrutura de cDNA de rhAPC foi buscada para permitir melhorotimização de códon com relação à célula de mamífero a ser usada em par-ticular. O design da estrutura de cDNA sintética também inclui característicastais como:
o Uma seqüência de consenso de Kozak (GCCACC), seguidopor um códon inicial (ATG) para assegurar tradução eficiente.
O Sítios de restrição adequados nas extremidades 5' e 3' docDNA para clonar no vetor de expressão desejado.
As seqüências de nucleotídeo da proteína C ativada humanaforam representadas em SEQ ID NQ: 1. Os códons na seqüência de DNA decodificação de rhAPC que foram alteradas como parte do processo de otimi-zação de códon para assegurar expressão ótima de proteína recombinanteem linhagens de células de mamífero, tais como CHO K1 e HEK 293. A se-qüência com códon otimizado do ácido nucléico foi representada em SEQ IDNQ: 2. A seqüência otimizada da seqüência de ácido nucléico foi representa-da em SEQ ID N9: 2.
Alinhamento de seqüência aos pares pós-otimização de códondas versões com códon otimizado e não-otimizado da seqüência de nucleo-tídeo de DNA que codifica Drotrecogina alfa ou Xigris foi representado naFigura 1.
Exemplo 2
Subclonaaem de cDNA de Drotrecoaina Alfa (DROT) no Vetor de ExpressãoCélula de Mamífero-Específico pcDNA3.1D/V5-His
Subseqüentemente à verificação da autenticidade das moléculasde cDNA sintetizadas de novo (DROT & DROT-Opt) através de seqüencia-mento automatizado de DNA conforme mostrado acima, DROT foi subclona-do no vetor de expressão de célula de mamífero-específico pcDNA3.1D/V5-His para gerar as estruturas prontas para transfecção. Os detalhes dos pro-cedimentos usados são fornecidos abaixo:A. Reaaentes e enzimas 1. Kit de extração em gel e kit de purificação por PCR da QIAGEN 2. DNA de Vetor pcDNA 3.1 D/V5-His (Invitrogen)
<table>table see original document page 7</column></row><table>
B. Diaestão por restrição do vetor e do inserto • Procedimento As amostras de DNA e enzimas de restrição a seguir foram usadas:
<table>table see original document page 7</column></row><table>
• Reação de digestão com enzima de restrição
<table>table see original document page 7</column></row><table>
A reação foi misturada, centrifugada e incubada durante 2 horasa 37°C. A digestão por restrição foi analisada através de eletroforese em gelde agarose. O padrão de digestão esperado foi observado. Um precipitadode fragmento de gene de -1400 bp (para Rxn Ne 2) e um fragmento de parteprincipal de vetor de -5,5 kb para o Vetor (Rxn Ns 1) foram observados. Osinsertos de -1400 bp do fragmento pcDNA3.1D/V5-His de vetor digerido deDROT de -5,5 kb foram purificados através de extração em gel usando o kitde extração em gel da QIAGEN. 1 μΙ do inserto purificado e fragmento devetor verificados sobre um gel de agarose a 1%.Os fragmentos digeridos por restrição purificados em gel de cD-NA de DROT e pcDNA3.1D/V5-His foram representados na Figura 2.
Exemplo 3
C. Ligação de parte principal de pcDNA3.1DA/5-His com cDNA de DROT
A concentração de DNA do vetor e fragmentos de inserto digeri-dos e purificados foi estimada (ref. Figura 7 acima) e a ligação foi ajustadada seguinte maneira:
<table>table see original document page 8</column></row><table>
As reações foram gentilmente misturadas, centrifugadas e incu-badas em R.T., 2-3 horas. Células DH10 competentes foram transformadascom as reações de ligação.
As colônias obtidas sobre lâminas de ágar LB contendo ampicili-na foram selecionadas e confirmadas através de análise através de digestãopor restrição do DNA de plasmídeo isolado.
Exemplo 4
D. Análise através de digestão por restrição de clones putativos de pcD-NA3.1DRO T-/V5-His/Xigrís
DNA de plasmídeo foi individualmente purificado a partir de co-lônias obtidas sobre lâminas de ágar LB contendo ampicilina e a presençado inserto de cDNA desejado foi confirmada por meio de análise através dedigestão por restrição do DNA de plasmídeo isolado foi conduzida. Análiseatravés de digestão por restrição de clones putativos de AVC1PpcDNA3,iD/v5-His/Xigris foi representada na Figura.
De acordo com os resultados obtidos após a digestão por restri-ção de vários clones putativos contendo o pcDNA3.1-DROT-D/V5-His/Xigris,alguns dos clones os quais mostraram o padrão de restrição por digestãoforam selecionados para análise através de digestão por restrição adicionalusando enzimas de restrição que clivam o cDNA de AVCIP-Xigris interna-mente para gerar fragmentos de tamanho variado conforme mostrado abaixona Figura 9.
Análise através de digestão por restrição de clones AVCiPpcD-NA3, iD/V5-His/Xigris usando enzimas que clivam cDNA de pcDNA3.1-DROT internamente.
A maioria dos clones pcDNA3.1 -DROT D/V5-His/Xigris selecio-nados para a análise de mapeamento por restrição proporcionaram os ta-manhos de fragmento esperados baseado sobre a ocorrência de sítios derestrição internos conhecidos e, conseqüentemente, esses clones foram ain-da verificados através de análise por seqüenciamento de DNA.
Exemplo 5
Verificação de autenticidade de moléculas de cDNA sintetizadas de novo
A verificação de autenticidade das moléculas de cDNA sintetiza-das de novo, conforme fornecido pelo fornecedor de serviço comercial, foifeita através de seqüenciamento de DNA automático.
E. Verificação de clones selecionados de ocDNA3.1 -DROT DA/5-His/Xigrisatravés de seqüenciamento de DNA
Os clones pcDNA3.1 -DROT D/V5-His/Xigris selecionados comoum resultado da análise de mapeamento por restrição foram ainda verifica-dos através de seqüenciamento automático de DNA.
<table>table see original document page 9</column></row><table>
O clone pcDNA3.1-DROT D/V5-His/Xigris mostrou identidadecom a seqüência DNA molde.
O mapa do DROT é Figuradamente representado na Figura 8.
Estrutura de expressão recombinante feita usando o pcDNA3.1- sintetizadode novo.Exemplo 6
Manutenção e propagação da estrutura de fusão rhAPC
A manutenção e propagação da estrutura de cDNA que codifica rhAPC foifeita em uma linhagem de célula bacteriana padrão, tal como Top 10 (Invitrogen).
Exemplo 7
Expressão de proteína recombinante transitória/estável em células HEK293e produção de sobrenadantes
a) Expressão transitória/estável da estrutura rhAPC foi feita u-sando as células de rim embriônico humano (HEK293), transformadas atra-vés de DNA do adenovírus do tipo 5 humano cisalhado (AD5), a qual é umalinhagem de célula de mamífero principal que é aprovada pelo FDA para a-plicações industriais. Expressão transitória é útil para verificar a expressãode uma estrutura e obter rapidamente pequenas quantidades de uma proteí-na recombinante.
b) Alternativamente, um protocolo que permitiu seleção de umagrande população de células que exibia alta expressão rapidamente, sem terde obter clones individuais. Subseqüentemente, células HEK293 que mos-traram uma expressão elevada e estável da proteína rhAPC desejada foramdesenvolvidas usando procedimentos padrões.
Técnicas de cultura aperfeiçoadas usando meios de cultura qui-micamente definidos (Sigma Aldrich) em oposição a meios contendo soroforam usadas durante todo o procedimento em conformidade com os requisi-tos do FDA.
Exemplo 8
Otimização de procedimentos de purificação
Subseqüentemente ao estabelecimento de bioatividade de acor-do com os ensaios funcionais/de ligação recomendados mencionados aci-ma, esforços serão feitos para otimizar os procedimentos de purificação demodo a maximizar o rendimento.
Conseqüentemente, o processo de purificação compreenderia aseguinte série a jusante:a. Clarificação e concentração iniciais usando procedimentos defiltração com fluxo tangencial e normal.
b. Ultra filtração/filtração por diálise (baseado em filtração comfluxo tangencial).
c. Etapa Cromo - I: cromatografia por afinidade usando anticor-po monoclonal ao sítio de ativação sobre a cadeia pesada de proteína C ati-vada ou um anticorpo cálcio-dependente dirigido ao domínio de ácido gamacarbóxi glutâmico da cadeia leve de proteína C humana.
d. Etapa Cromo - II: cromatografia por troca de ânions usandofractogel EMD.
e. Etapa Cromo - III: Escoamento baseado em permutadores deânions, tal como sulfato de cellufine, para a remoção de DNA e proteínas decélulas hospedeiras.
f. Remoção de vírus e filtração estéril.
g. Remoção de endotoxina.
h. Formulação.
LISTAGEM DE SEQÜÊNCIA XIGRIS
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gcc ttc tgg tcc aag cac gtc gac ggt gac cag tgc ttg gtc ttg ccc 288Ala Phe Trp ser Lys His vai Asp Gly Asp Gln cys Leu Val Leu pro85 90 95
ttg gag cac ccg tgc gcc age ctg tgc tgc ggg cac ggc acg tgc ate 336Leu Glu His Pro cys Ala ser Leu Cys cys Gly His Gly Thr Cys Ile100 105 110
gac ggc ate ggc age ttc age tgc gac tgc cgc age ggc tgg gag ggc 384Asp Gly ile Gly ser phe ser cys Asp cys Arg ser Gly Trp Giu Giy115 120 125
cgc ttc tgc cag cgc gag gtg age ttc ctc aat tgc tcg ctg gac aac 432Arg Phe Cys Gln Arg Glu VaT ser Phe Leu Asn Cys Ser Leu Asp Asn130 135 140
ggc ggc tgc acg cat tac tgc cta gag gag gtg ggc tgg cgg cgc tgtGly Gly cys Thr His Tyr Cys Leu Glu Glu vai Gly Trp Arg Arg cys145 150 155 160
age tgt gcg cct ggc tac aag ctg ggg gac gac ctc ctg cag tgt cacSer cys Ala pro Gl^ Tyr Lys Leu Gly Asg Asp Leu Leu Gln His
ctg ctg gac tca aag aag aag ctg gcc tgc ggg gea gtg ctc ate cacLeu Leu Asp ser Lys Lys Lys Leu Ala Cys Gly Ala vai Leu tle His225 230 235 240
ctt gea gag cgc gag ctc aat cag gcc ggc cag gag acc ctc gtg acgLeu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu Tbr Leu VaT Thr325 330 335
480
528
ccc gea gtg aag ttc cct tgt ggg agg ccc tgg aag cgg atg gag aag 576Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Trp Lys Arg Met Glu Lys180 185 190
aag cgc agt cac ctg aaa cga cct cga ccg tcc cgg aaa cgc agg ctc 624Lys Arg ser His Leu Lys Arg Pro Arg Pro ser Arg Lys Arg Arg Leu195 200 205
atrt gat ggg aag atg acc agg cgg gga gac age ccc tgg cag gtg gtc 672Ile Asp Gly Lys Met Thr Arg Arg Giy Asp ser Pro Trp Gln VaT vai210 215 220
720
ccc tcc tgg gtg ctg aca gcg gcc cac tgc atg gat gag tcc aag aag 768Pro Ser Trp VaT Leu Thr Ala Ala His cys Met Asp Glu Ser Lys Lys245 250 255
ctc ctt gtc agg ctt gga gag tat gac ctg cgg cgc tgg gag aag tgg 816Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp260 265 270
gag ctg gac ctg gac ate aag gag gtc ttc gtc cac ccc aac tac age 864Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His Pro Asn Tyr ser275 280 285
aag age acc acc gac aat gac ate gea ctg ctg cac ctg gcc cag ccc 912
Lys Ser Thr Thr Asp Asn Asp lie Ala Leu Leu His Leu Ala Gln Pro
290 295 300
gcc acc ctc tcg cag acc ata gtg ccc ate tgc ctc ccg gac age ggc 960Ala Thr Leu ser Gln Thr Xle vai Pro lie cys Leu Pro Asp ser Gly305 310 315 320
1008
ggc tgg ggc tac cac age age cga gag aag gag gcc aag aga aac cgc 1056Gly Trp Gly Tyr His ser ser Arg Glu Lys Glu Ala Lys Arg Asn Arg340 345 350
acc ttc gtc ctc aac ttc ate aag att ccc gtg gtc ccg cac aat gag 1104Thr Phe Val Leu Asn Phe Ile Lys Ile Pro vai Val Pro His Asn Glu355 360 365
tgc age gag gtc atg age aac atg gtg tet gag aac atg ctg tgt gcg 1152Cys Ser Glu vai Met ser Asn Met vai ser Glu Asn Met Leu Cys Ala370 375 380
ggc ate ctc ggg gac cgg cag gat gcc tgc gag ggc gac agt ggg ggg 1200Gly Ile Leu Gly Asp Arg Gln Asp Ala Cys Glu Gly Asp ser Gly Gly385 390 395 400
ccc atg gtc gcc tcc ttc cac ggc acc tgg ttc ctg gtg ggc ctg gtgPro Met Val Ala ser Phe His Gly Thr Trp Phe Leu VaT Gly Leu Vai405 410 415
age tgg ggt gag ggc tgt ggg ctc ctt cac aac tac ggc gtrt tac accSer Trp Gly Glu Gly cys Gly Leu Leu His Asn Tyr Gly Val Tyr Thr420 425 430
1248
1296
aaa gtc age cgc tac ctc gac tgg ate cat ggg cac ate aga gac aag 1344Lys Val Ser Arg Tyr Leu Asp Trp Ile His Gly His Ile Arg Asp Lys435 440 445
gaa gcc ccc cag aag age tgg gea cct tag 1374
Glu Ala Pro Gln Lys ser Trp Ala Pro450 455
<210> 2
<211> 457
<212> PRT
<213> Homosapiens
<400> 2
Met Trp Gln Leu Thr ser Leu Leu Leu Phe vai Ala Thr Trp Gly Ile1 5 10 15
ser Gly Thr Pro Ala Pro Leu Asp ser vai Phe ser ser ser Glu Arg20 25 30
Ala His Gln vai Leu Arg lie Arg Lys Arg Ala Asn ser Phe Leu Glu35 40 45
Glu Leu Arg His ser ser Leu Glu Arg Glu cys Ile Glu Glu He cys50 55 60
Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln Asn Val Asp Asp Tlir Leu65 70 75 80
Ala Phe Trp ser Lys His vai Asp Gly Asp Gln cys Leu Val Leu PrO85 90 95
Leu Glu His Pro Cys Ala ser Leu Cys Cys Gly His Gly Ttir cys Ile100 105 110
Asp Gly Ile Gly ser Phe ser cys Asp cys Arg ser Gly Trp Glu Gly115 120 125
Arg Phe cys Gln Arg Glu Val ser Phe Leu Asn Cys Ser Leu Asp Asn130 135 140Gly Gly cys Thr His Tyr Cys Leu Glu Glu vai GTy Trp Arg Arg Cys145 150 155 160
ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp Asp Leu Leu Gln cys His
165
170
175
Pro Ala vai Lys Phe Pro cys Gly Arg Pro Trp Lys Arg Met Glu Lys180 185 190
Lys Arg ser His Leu Lys Arg Pro Arg Pro ser Arg Lys Arg Arg Leu195 200 205
Ile Asp Gly Lys Met Thr Arg Arg Gly Asp ser pro Trp Gln vai Val210 215 220
Leu Leu Aso ser Lys Lys Lys Lsu Ala cys Sly Ala Val Leu He His225 2^0 235 240
Pro ser Trp vai Leu Thr Ala Ala His cys Met Asp Glu ser Lys Lys245 250 255
Leu Leu vai Arg Leu Gly Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp260 265 270
Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His Pro Asn Tyr ser275 280 285
Lys ser Thr Thr Asp Asn Asp Ile Ala Leu Leu His Leu Ala Gln Pro290 295 300
Ala Thr Leu Ser Gln Thr Ile Val Pro Ile cys Leu Pro Asp ser Gly305 310 315 320
Leu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu Thr Leu Val Thr325 330 335
Gly Trp Gly Tyr His Ser Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg340 345 350
Thr Phe vai Leu Asn Phe Ile Lys Π e Pro Val Val Pro His Asn Glu
355
360
365
cys Ser Glu Val Met Ser Asn Met Val ser Glu Asn Met Leu cys Ala
370
375
380
Gly Ile Leu Gly Asp Arg Gln Asp Ala cys Glu Gly Asp ser Gly Gly385 390 395 400Pro Het Val Ala Ser Phe His Gly Thr Trp Phe Leu Val Gly Leu Val405 410 415
Ser Trp Gly Glu Gly cys Gly Leu Leu His Asn Tyr Gly Val Tyr Thr420 425 430
Lys Val ser Arg Tyr Leu Asp Trp Xle His Gly His Xle Arg Asp Lys435 440 445
Glu Ala Pro Gln Lys Ser Trp Ala Pro450 455
<210> 3<211> 457<212> PRT<213> Homosapiens<220>
<221 > PEPTÍDEO<222> (1)...(457)<400> 3
Met Trp Gln Leu Thr ser Leu Leu Leu Phe Val Ala Thr Trp Gly Xle1 5 10 15
Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Phe Ser Ser ser Glu Arg20 25 30
Ala His Gln vai Leu Arg lie Arg Lys Arg Ala Asn Ser Phe Leu Glu35 40 45
Glu Leu Arg His ser ser Leu Glu Arg Glu Cys Ile Glu Glu Ile Cys50 55 60
Asp Phe Glu Glu Ala Lys Glu lie Phe Gln Asn Val Asp Asp Thr Leu65 70 75 80
Ala Phe Trp ser Lys His vai Asp Gly Asp Gln cys Leu vai Leu pro85 90 95
Leu Glu His Pro cys Ala ser Leu cys cys Gly His Gly Thr cys Ile100 105 110
Asp Gly Ile Gly ser Phe ser cys Asp cys Arg ser Gly Trp Glu Gly115 120 125
Arg Phe cys Gln Arg Glu Val ser Phe Leu Asn Cys ser Leu Asp Asn130 135 140Gly Gly cys Thr His Tyr Cys Leu Glu Glu Val Gly Trp Arg Arg Cys145 ΐ!θ 155 160
Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp Asp Leu Leu Gln Cys His165 170 175
Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Trp Lys Arg Met Glu Lys180 185 190
Lys Arg ser His Leu Lys Arg Pro Arg Pro Ser Arg L^s Arg Arg Leu
195
200
Ile Asp Gly Lys Met Thr Arg Arg Gly Asp ser Pro Trp Gln Val Val210 215 220
Leu Leu Asp ser Lys Lys Lys Leu Ala Cys Gly Ala Val Leu Ile His225 230 235 240
Pro ser Trp Val Leu Thr Ala Ala His cys Met Asp Glu ser Lys Lys245 250 255
Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp260 265 270
Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His Pro Asn Tyr ser
27
280
285
Lys ser Thr Thr Asp Asn Asp Ile Ala Leu Leu His Leu Ala Gln Pro
290
295
300
Ala Thr Leu ser Gln Thr Ile vai Pro lie cys Leu Pro Asp ser Gly305 310 315 320
Leu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu Thr Leu vai Thr325 330 335
Gly Trp Gly Tyr His ser ser Arg Glu Lys Glu Ala Lys Arg Asn Arg340 345 350
Thr Phe Val Leu Asn Phe Ile Lys Ile Pro Val Val Pro His Asn Glu355 360 365
Cys ser Glu Val Met ser Asn Met Val ser Glu Asn Met Leu cys Ala370 375 380
Gly He Leu Gly Asp Arg Gln Asp Ala cys Glu Gly Asp Ser Gly Gly385
ccc atg gtcρpo Met vai
tcc tgg ggcser Trp Giy
aag gtt tcaUjrs Val ser435
ges gçt cccGiu Aia pra450
390
395
400
gcc tct ttc cac ggc acc tgg ttc tta gtg gga dtg gtgAla ser Phe His ely Thr Trp Phe Eeu vai Gly Ljeu vai
405
Qaa ggcGiu Gly420
410
415
tgc ggg ctg ctc cac aac tac ggc gtg tac acccys Gly Leu Leu His asii Tyr Gly vai Tyr Thr425 430
cgc tac ctç gat tgg cat gga ftac ate cgc gac aaaArg Tyr LeL1 Asp Trp He His Giy Bis Ilc Arg asp Lys
440 445
cag aag tcC tgg gcc ccc tagGln Lys Sei? Trp Ala ppo
455
12481296
13441374
Claims (6)
1. Processo para o preparo de produto de proteína C humanaativada biologicamente in vivo compreendendo etapas de transformação deuma célula hospedeira com uma seqüência de DNA sintetizada que codificaa proteína codificada pela seqüência de ácido nucléico de SEQ ID N5: 2 eisolamento do referido produto da referida célula hospedeira ou do meio deseu crescimento.
2. Método de acordo com a reivindicação 1, em que a seqüênciade ácido nucléico com códon otimizado que codifica a proteína C humanaativada foi representada em SEQ ID Ne: 3.
3. Processo de acordo com a reivindicação 1, em que as célulashospedeiras são células de mamífero.
4. Processo de acordo com a reivindicação 1, em que as célulashospedeiras são, de preferência, selecionadas da cepa HEK293.
5. Processo para o preparo de um produto de proteína C ativadarecombinante humana biologicamente ativa in vivo compreendendo as eta-pas de transformação de uma célula hospedeira com uma estrutura de vetorda Figura N- 8 e isolamento do referido produto da referida célula hospedei-ra ou do meio de seu crescimento.
6. Processo de acordo com a reivindicação 1, em que o referidovetor é um vetor de expressão específico para células de mamífero e, maispreferivelmente, um vetor conforme representado na Figura N6 8.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN626CH2005 | 2005-05-24 | ||
| IN626/CHE/2005 | 2005-05-24 | ||
| PCT/IB2006/001359 WO2006126070A2 (en) | 2005-05-24 | 2006-05-24 | A process comprising codon optimization for the production of recombinant activated human protein c for the treatment of sepsis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| BRPI0611376A2 true BRPI0611376A2 (pt) | 2010-08-31 |
Family
ID=37452412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| BRPI0611376-1A BRPI0611376A2 (pt) | 2005-05-24 | 2006-05-24 | processo para a producão de proteìna c ativada humana para o tratamento de sepsia |
Country Status (14)
| Country | Link |
|---|---|
| US (1) | US20090068721A1 (pt) |
| EP (1) | EP1888744A2 (pt) |
| JP (1) | JP2009502118A (pt) |
| KR (1) | KR20080021682A (pt) |
| CN (1) | CN101228269A (pt) |
| AP (1) | AP2007004253A0 (pt) |
| AU (1) | AU2006250889A1 (pt) |
| BR (1) | BRPI0611376A2 (pt) |
| CA (1) | CA2609435A1 (pt) |
| IL (1) | IL187477A0 (pt) |
| MX (1) | MX2007014674A (pt) |
| RU (1) | RU2007147432A (pt) |
| WO (1) | WO2006126070A2 (pt) |
| ZA (1) | ZA200711006B (pt) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014202573A1 (en) * | 2013-06-17 | 2014-12-24 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for modulating gene expression |
| WO2019199808A1 (en) * | 2018-04-09 | 2019-10-17 | The Wistar Institute | Engineered optimized cytokine compositions |
| KR20250000042A (ko) | 2023-06-23 | 2025-01-02 | 충남대학교산학협력단 | 절단 반응성 펩타이드를 발현하는 키메라 t 세포 및 이를 포함하는 감염 질환 치료용 조성물 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3688900T3 (de) * | 1985-06-27 | 1998-06-10 | Univ Washington | Expression von Protein C. |
| US4992373A (en) * | 1987-12-04 | 1991-02-12 | Eli Lilly And Company | Vectors and compounds for direct expression of activated human protein C |
| US5358932A (en) * | 1989-12-29 | 1994-10-25 | Zymogenetics, Inc. | Hybrid protein C |
| EP1272508A2 (en) * | 1999-09-21 | 2003-01-08 | Prodigene Inc. | Methods for producing recombinant proteins |
-
2006
- 2006-05-24 BR BRPI0611376-1A patent/BRPI0611376A2/pt not_active Application Discontinuation
- 2006-05-24 AP AP2007004253A patent/AP2007004253A0/xx unknown
- 2006-05-24 JP JP2008512944A patent/JP2009502118A/ja active Pending
- 2006-05-24 US US11/914,751 patent/US20090068721A1/en not_active Abandoned
- 2006-05-24 CA CA002609435A patent/CA2609435A1/en not_active Abandoned
- 2006-05-24 AU AU2006250889A patent/AU2006250889A1/en not_active Abandoned
- 2006-05-24 KR KR1020077029877A patent/KR20080021682A/ko not_active Withdrawn
- 2006-05-24 EP EP06744762A patent/EP1888744A2/en not_active Withdrawn
- 2006-05-24 MX MX2007014674A patent/MX2007014674A/es not_active Application Discontinuation
- 2006-05-24 CN CNA2006800269242A patent/CN101228269A/zh active Pending
- 2006-05-24 RU RU2007147432/13A patent/RU2007147432A/ru not_active Application Discontinuation
- 2006-05-24 WO PCT/IB2006/001359 patent/WO2006126070A2/en not_active Ceased
-
2007
- 2007-11-19 IL IL187477A patent/IL187477A0/en unknown
- 2007-12-19 ZA ZA200711006A patent/ZA200711006B/xx unknown
Also Published As
| Publication number | Publication date |
|---|---|
| MX2007014674A (es) | 2008-03-07 |
| CN101228269A (zh) | 2008-07-23 |
| WO2006126070A2 (en) | 2006-11-30 |
| AP2007004253A0 (en) | 2007-12-31 |
| WO2006126070A3 (en) | 2007-04-12 |
| US20090068721A1 (en) | 2009-03-12 |
| ZA200711006B (en) | 2008-11-26 |
| CA2609435A1 (en) | 2006-11-30 |
| JP2009502118A (ja) | 2009-01-29 |
| RU2007147432A (ru) | 2009-06-27 |
| EP1888744A2 (en) | 2008-02-20 |
| AU2006250889A1 (en) | 2006-11-30 |
| IL187477A0 (en) | 2008-03-20 |
| KR20080021682A (ko) | 2008-03-07 |
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