FI91280B - Repressibla jästpromotorer - Google Patents
Repressibla jästpromotorer Download PDFInfo
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- FI91280B FI91280B FI863430A FI863430A FI91280B FI 91280 B FI91280 B FI 91280B FI 863430 A FI863430 A FI 863430A FI 863430 A FI863430 A FI 863430A FI 91280 B FI91280 B FI 91280B
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/14—Hydrolases (3)
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Claims (12)
1. En uppströms-aktiveringssekvens, känneteck-n a d därav, att den innefattar en del av jästens PH05-gen, och att det är fräga om uppströms-aktiveringssekven-sen UAS1 (PH05) eller UAS2 (PH05), son ligger i BamHI-BstEII-fragment i jästens PHOS-gen mellan nukleotiderna -174 och -541, varvid uppströms-aktiveringssekvensen UASl (PH05), ligger i BamHI-Clal-fragment av jästens PH05-gen mellan nukleotiderna -274 och -541, och mera specifikt i DNA-fragmentet med sekvensen GAAATATATATTAAATTAGCACGTTTTCGCA CTTTATATATAATTTAATCGTGCAAAAGCGT och uppströms-aktiveringssekvensen UAS2 (PH05) ligger i Clal-BstEII-fragmentet i jästens PH05-gen mellan nukleotiderna -174 och -273.
2. Jäst-hybridpromotor, kännetecknad därav, att den innehäller ett 51-uppströms-promotorelement, son har uppströms-aktiveringsstället(na) av jästens PH05-gen enligt patentkravet 1, och ett 3'-nedströms-promotorele-ment av jästens GAPDH-gen, som är ett DNA-fragment mellan -5 och -199 av jästens GAPDH-promotor eller ett DNA-fragment mellan -5 och -263 av jästens GAPDH-promotor.
3. Hybridpromotor enligt patentkravet 2, kännetecknad därav, att 5'-uppströms-promotorelementet är ett 368 bp BamHI-BstEII-fragment av 5'-regionen av jästens PH05-gen, ett 268 bp BamHI-Clal-fragment av 5'-regionen av jästens PH05-gen, ett 31 bp fragment med forme In GAAATATATATTAAATTAGCACGTTTTCGCA CTTTATATATAATTTAATCGTGCAAAAGCGT 91280 eller ett 100 bp Clal-BstEII-fragment av 5'-regionen av jästens PH05-gen.
4. Hybridpromotor enligt patentkravet 2, kanne- t e c k n a d därav, att den innehäller UAS1 (PH05) och/ eller UAS2 (PH05).
5. Hybridpromotor enligt patentkravet 2, kanne- t e c k n a d därav, att 31-nedströms-promotorelementet är härlett frän promotorn av en jästgen, som kodar för ett glykolytiskt enzym.
6. Jäst-hybridvektor, kännetecknad därav, att den innehäller en eller flera inklonade DNA-sekven-ser, som var och en har ett DNA segment, som kodar för en polypeptid heterolog tili jäst i transkriptionell kont-roll av en sädan hybridpromotor, som bestär av ett 5'-uppströms-promotorelement, som har UAS(r) av jästens PH05-gen, och av ett 3'-nedströms-promotorelement, som är ett DNA-fragment mellan -5 och -199 av jästens GAPDH-pro-motor eller ett DNA-fragment mellan -5 och -263 av jästens GAPDH-promotor.
7. Jäst-hybridvektor enligt patentkravet 6, kännetecknad därav, att den innehäller ett 5'-upp-ströms-promotorelement antingen enligt patentkravet 3 eller 4 och ett 3'-nedströms-promotorelement enligt patentkravet 5.
8. Jäst-hybridvektor enligt patentkravet 6, kännetecknad därav, att den innehäller ett DNA-segment, som kodar för en polypeptid av högre eukaryotiskt ur-sprung.
9. En transformerad jästvärd, kännetecknad därav, att transformeringen utförts med en hybridvektor, som innehäller en eller flera inklonade DNA-sekvenser, som var och en har ett DNA-segment, som kodar för en po-lypeptid heterolog tili jäst i transkriptionell kontroll av en sädan hybridpromotor, som bestär av ett 5'-upp-ströms-promotorelement, som har UAS(r) av jästens PH05-gen enligt patentkravet 1, och av ett 3'-nedströms-promo-torelement av GAPDH-gen, som innehäller transkriptions-initiatorsställen inberäknad den funktionella TATA-regio-nen.
10. Förfarande för produktion av en tili jäst heterolog polypeptid, kännetecknat därav, att man od-lar en jäststam, som transformerats med en hybridvektor, som innehäller en eller flera inklonade DNA-sekvenser, som var och en har ett DNA segment, som kodar för en po-lypeptid heterolog tili jäst i transkriptionell kontroll av en sädan hybridpromotor, som bestär av ett 5'-upp-ströms-promotorelement, som har UAS(r) av jästens PH05-gen enligt patentkravet 1, och av ett 31-nedströms-promo-torelement av GAPDH-gen, som innehäller transkriptions-initiatorsställen inberäknad den funktionella TATA-regio-nen, och tillvaratar den uttryckta polypeptiden.
11. Förfarande enligt patentkravet 10, kännetecknat därav, att DNA-sekvensen kodar för en po-lypeptid av högre eukaryotiskt ursprung.
12. Förfarande enligt patentkravet 10, kännetecknat därav, att polypeptiden, som kodas av DNA-sekvensen, är human vävnads-plasminogenaktivator (t-PA), desulfatohirudin, eglin C eller insulin-liknande växtfak-tor.
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ATE85812T1 (de) * | 1985-11-13 | 1993-03-15 | Xoma Corp | Verkuerzter phosphoglycerat-kinase-promotor. |
GB8530631D0 (en) | 1985-12-12 | 1986-01-22 | Ciba Geigy Ag | Thrombin inhibitors |
MY101203A (en) * | 1985-12-12 | 1991-08-17 | Ucp Gen Pharma Ag | Production of thrombin iinhibitors. |
GB8620926D0 (en) * | 1986-08-29 | 1986-10-08 | Delta Biotechnology Ltd | Yeast promoter |
GB8907110D0 (en) * | 1988-05-04 | 1989-05-10 | Ciba Geigy Ag | Improvements in the production of polypeptides |
NO177065C (no) * | 1988-09-26 | 1995-07-12 | Labofina Sa | Framgangsmåte for framstilling av enzymatisk aktivt humant lysozym |
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JPH08173170A (ja) * | 1994-05-25 | 1996-07-09 | Kirin Brewery Co Ltd | キャンディダ・ユティリス酵母の形質転換系、およびそれによる異種遺伝子の発現 |
WO1998013513A2 (en) * | 1996-09-24 | 1998-04-02 | Cadus Pharmaceutical Corporation | Methods and compositions for identifying receptor effectors |
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US6890730B1 (en) | 1999-12-10 | 2005-05-10 | The Salk Institute | Sequence and method for increasing protein expression in cellular expression systems |
US8008459B2 (en) * | 2001-01-25 | 2011-08-30 | Evolva Sa | Concatemers of differentially expressed multiple genes |
WO2011146833A1 (en) | 2010-05-20 | 2011-11-24 | Evolva Inc. | Method of producing isoprenoid compounds in yeast |
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WO2023122805A1 (en) | 2021-12-20 | 2023-06-29 | Vestaron Corporation | Sorbitol driven selection pressure method |
TW202413392A (zh) | 2022-05-18 | 2024-04-01 | 美商韋斯塔隆公司 | 殺蟲actx肽變異體 |
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GB2125047B (en) * | 1982-08-09 | 1986-02-19 | Ciba Geigy Ag | Yeast hybrid vectors and their use for the production of polypeptides |
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NZ207926A (en) * | 1983-04-25 | 1988-04-29 | Genentech Inc | Use of yeast #a#-factor to assist in expression of proteins heterologus to yeast |
EP0127839B1 (en) * | 1983-05-27 | 1992-07-15 | THE TEXAS A&M UNIVERSITY SYSTEM | Method for producing a recombinant baculovirus expression vector |
GB8314961D0 (en) * | 1983-05-31 | 1983-07-06 | Kingsman A J | Dna sequence |
PT79518B (pt) * | 1983-11-21 | 1986-12-12 | Ciba Geigy Ag | 54). 5 ^ig do plasmídeo pBR 322 /PHO5 Bam Rsa 637 são digeridos com a endonuclease de restrição Xbal. Apos digestão completa o DNA ê extraído com fenol/clorofórmio e precipitado com etanol. 0 DNA ê redissolvido em Tris.HCl 50mM pH8,0 numa concentração de 50 p-g/nd. e ê digerido com 11 unidades de fosfatase alcalina do intestino de vitela (Boehringer), durante 1 hora a 379C. - 97 fORHjf y| A enzima ê inativada a 65 9C durante 1 hora. 0 DNA ê purificado por cromatografia de troca iõnica em DE 52 ( Whatman) e precipitação com etanol como descrito no Exemplo 9Aa. Dois oligodeoxinucleotídeos sintéticos com a formula 5’-CTAGAAGGGGTATCTTTGGATAAGA - 3' 3’- TTCCCCATAGAAACCTATTCTCTAG -5' são fosforilados individualmente nas suas extremidades 5' como descrito no Exemplo 9Ab. Os oligodeoxinacleotídeos fosforilados são misturados em quantidades equimolares . A mistura ê aquecida durante 10 min. a 759C, depois deixada arrefecer lentamente atê à temperatura ambiente e guardada a -209C. Este adaptador ê designado por adaptador C. 1 ^ig (120 pmoles) do adaptador C emparelhado e fosforilado e 5 pg (1,5 pmoles) de pBR322/ /PH05 Bam-Rsa 637 desfosforilado e cortado com Xba I são ligados em 40^1 de Tris.HCl 60mM pH 7,5, MgCl2 lOmM, DTT 5mM, ATP lmM, 1600 unidades da ligase de DNA do T4 a 159C durante 20 horas. A ligase de DNA ê inactivada durante 10 min. a 859C e o excesso de adaptadores ê removido por precipitação com isopropanol. 0 DNA ê redissolvido em H20 numa concentração de 0,25 mg/ml. Os múltiplos de adaptadores são removidos por digestão com a endonuclease de restrição BglII. 0 DNA ê precipitado com etanol, redissolvido e depois digerido com Bam HI. Os fragmentos de restrição são separados num gel de 0,6¾ de agarose de baixo ponto de fusão em Tris-borato-EDTA pH8,3. 0 fragmento Bam HI-BglII de 662 pb (derivado de fragmento Bam HI-Rsa I de 637 pb) ê localizado e removido do gel. c) Isolamento de um fragmento de vector contendo a sequência codificadora do TPA maduro ( fig. 23) 5 yig do plasmídeo p31R/SS-TPA /\ 2, são digeridos com as endonucleases de restrição Bam HI e BglII. Após digestão completo de DNA e precipitado com etanol e ressuspenso em Tris.HCl 50mM pH 8,0 numa concentração de 75 jxg /ml. Os fragmentos de DNA sao desforilados nas suas extremidades 5' por digestão com 1 unidade de fosfatase alcalina de intestino de vitela ( Boehringer) em 60 de Tris.HCl 50mM pH 8,0 durante 1 hora a 379 C. A fosfatase ê inactivada por incubação a 659C durante 1,5 horas. 0 DNA é purificado por cromatografia de troca iónica em DE-52 (Whatman) como descrito no Exemplo 9Aa. 0 DNA ê precipitado com etanol, redissolvido em Tris-HCl lOmM, pH8,0 e aplicada num gel de 0,6 °'o de agarose de ponto de fusão baixo. 0 fragmento maior Bam HI -BglII de 5,2 kb ê removido do gel. d) Ligação dos fragmentos de DNA isolados e transformação de E.coli HB 101 (cf. figs 23 e 24 ). Dois blocos de gel de agarose de ponto de fusão baixo contendo 0,1 pmoles do fragmento Bam HIBglII de 5,2 kb de p31R/SS-TPA/\ 2 e 0,2 pmoles do fragmen to BamHI -BglII de 662 pb de pBR322/PHO5 Bam-Rsa 637, respectivamente, são misturados, liquefeitos a 659C e diluídos para baixar a concentração de agarose para 0,3¾. A ligação ê feita em 150^1 de Tris.HCl 60mM, pH 7,5; MgCl^ lOmM, 99 jr £ DTT 5mM, ATP ImM e 800 unidades de ligase de DNA do T4 (Biolabs) a 159C durante 16 horas. Amostras de 10 ^il e 30 yjl são misturadas com 100 p.1 de células E.coliHB 101 competentes para a transformação como descrito no Exemplo 4a. - R 6 colonias transformadas amp são crescidas individualmente em meio LB contendo 100 ^g/ml de ampicilina. 0 DNA de plasmídeo ê analisado quanto ao tamanho e orientação da invenção por corte com a endonuclease de restrição Bam Hl. Um único clone com a orientação correcta da inserção é designado por p31/PHO5 637-TPA (cf. fig. 23). 0 plasmídeo pBR 322/PHO5 Bam-Rsa 637 pode também ser substituído por pBR 322/PHO5 Bam-Rsa 595, pBR 322/PHO5 Bam-Rsa 811 e pBR 322/PHO5 Bam-Rsa 1312 . Seguindo o processo descrito atras são isolados clones únicos com inserções do tamanho e orientação esperados e designados por p31/PHO5 595C-TPA, p51/PHO5 811C-TPA e p31/PHO5 1312 C-TPA. 0 adaptador C pode também ser substituído pelo adaptador B com a seguinte férmula 5' -CTAGATAAGAGATCTGC -3’ 3’ TATTCTCTAGACG -5' o qual codifica Lis -Arg na construção final (fig. 24). A fosforilação dos oligodeoxinucleotídeos sintéticos e emparelhamento são como descrito para o adaptador C. Para a adição do adaptador 5 de pBR 322/PHO5 Bam-Rsa 637 cortado com Xba I são digeridos com 2 unidades de nuclease (Sigma) em 50 yjl de acetato de sédio 30mM pH 4,6, NaCI 200mM e ZnSO^ ImM durante 40 min. a 379C para criar extremidades cerses (ver fig.23). Apos extração com fenol clorofórmio o DNA ê precipitado com etanol e redissolvido em H^O numa concentração de 0,25 mg/ml. 0,4 ^g (120 pmoles) de adaptador A fosforilado e emparelhado e 5 ^ig (1,5 pmoles) de pBR 322/PHO5 Bam -Rsa 637 cortado com Xba I /S^ são ligados através das extremidades cerses como descrito para o adaptador C exceptuando a utilização de ATP 3,5 mM. As outras construções são como descrito atras. Os clones resultantes são identificado pela letra A e referidos como p31/PHO5 595A-TPA, p31/PHO5 637A-TPA, p31/PHO5 811A-TPA e p31/PHO5 1312 A-TPA. Exemplo 16: Subclonagem de construção genética no vector de levedura de elevado numero de copias pJDB2O7 e transformação de S. cerevisiae GRF18 As construções descritas nos Exemplos 13-19 contêm o promotor de PH05 com ou sem extensões para a região codificadora do gene de PH05 . A região codificadora do TPA com ou sem prepo-sequência e os sinais de terminação da transcrição de PH05 num arranjo tendem, tudo inserido num vector derivado do pBR 322. Os fragmentos Bam HI-HIND III contendo todo o arranjo são ligados ao fragmento Bam Hl -Hind III de 6,4 kb do pJDB 207 como descrito para uma reacção analoga no Exemplo 10. Células E.coli HB101 competentes são transformadas e vãrias colonias amp^ de cada experiência são crescidas individualmente em meio LB com 100 ^ig/ml de ampicilina. 0 DNA de plasmídio é analisado por corte com as endonucleases de restrição Hind III, Bam Hl e Pst I. Partindo dos plasmídeos p31/PHO5-TPA 18, ρ31 /PHO5-TPA 42, p31R/SS-TPA /\ 2, p31R/SS-TPA/\ 3, Ρ31/ΡΗΟ5 595C-TPA, p31/PHO5 637C-TPA,’ p31/PHO5 811C-TPA, P31/PHO5 1312C-TPA, p31/PHO5 595B-TPA, p31/PHO5 637-TPA, P31/PHO5 811 B-TPA, p31/PHO5 1312 B-TPA, p31/PHO5 595 A-TPA P31/PHO5 637-TPA, p31/PHO5 811-A-TPA e p31/PHO5 1312A-TPA obtem-se os clones que se seguem com as inserções correctas PJDB2O7/PHO5-TPA18 PJDB2O7/PHO5-TPA42 PJDB2O7R/PHO5-SS-TPA Λ 2 PJDB2O7R/PHO5-SS-TPA Λ 3 PJDB2O7/PHO5 595C-TPA PJDB2O7/PHO5 637C-TPA PJDB2O7/PHO5 811C-TPA PJDB2O7/PHO5 1312C-TPA PJDB2O7/PHO5 595B-TPA PJDB207/PH05 637B-TPA pJDB2O7/PHO5 811B-TPA PJDB2O7/PHO5 1312B-TPA pJDB2O7/PHO5 595A-TPA PJDB2O7/PHO5 637A-TPA PJDB2O7/PHO5 811A-TPA PJDB2O7/PHO5 1312A-TPA . Estes plasmídeos são introduzidos individualmente em Saccharomyces cerevisiae estirpe GRF 18 como descrito no Exemplo 11. Uma única colónia de levedura de cada uma das transformações de levedura ê picked. Os clones obtidos são designados por . Saccharomyces cerevisíae GRF18/pJDB2Ò7/PHO5-TPA18 Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5-TPA42 Saccharomyces cerevisíae GRF18/pJDB2O7R/PHO5-SS-TPA/\ 2 Sacchãtúiuycea cerevisíae GRFi8/pJD32O7R/?HO5~SS-TPA /\ 3 Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 595C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 637C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 811C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 1312C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 595B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 637B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 811B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 1312B-TPA As células de levedura são crescidas colhidas e extraídas como descrito nos Exemplos 11 e 12. A actividade de TPA nos extractos celulares é testado como descrito no Exemplo 12. Os resultados estão apresen tados na tabela 3. Tabela 3: Actividade de TPA da estirpe de levedura Saccharomyces cerevisiae GRF18 transformada com diferentes plasmídeos e testada em condições de desrepressão (Pi baixo) : plasmídeo Actividade de TPA(unidades/l de cultura de células de levedura/ OD^qq) PJDB2O7/PHO5-TPA18 750 PJDB2O7/PHO5-TPA42 700 PJDB2O7R/PHO5-SS-TPA_A 2 600 PJDB2O7R/PHO5-SS-TPA 3 650 PJDB2O7/PHO5 595C-TPA 900 Exemplo 17: Substituição cromossómica do gene de PHO5 pelo gene do TPA (ver fig.25) A região codificadora da proteína TPA ê colocada no cromossoma II de levedura na posição da região codificadora da proteína PH05 . Experimentálmente isto ê con seguido por cotransformação. Saccharomyces cerevisiae estirpe GRF 18 ê cotransformada com 25 ^ig do plasmídeo p31/PHO5 -TPA 18, linearizado com BnmHI e Hind III, e 3 ^xg do plasmídeo de levedura Yepl3 (51). De 24 colónias Leu obtidas, uma ê Pho5 e ao mesmo tempo produz aproximadamente 35 unidades de TPA/1 de cultura de células de levedura /0D600. Por crescimento deste transformante num meio contendo leucina (YPD, 10g/l de Bacto Yeast Extract, 20g/l de Bacto Peptone, 20 g/l de glucose) durante 10 gerações, segregantes Leu~são obtidos com uma frequência de 10-20¾ os quais provavelmente perderam o plasmideo Yep31. Por análise ou Southern do DNA total de levedura isolado dos segregantes Leu (52) verifica-se que no nível de DNA que as sequências codificadoras da proteína TPA substituem as sequências codificadores da proteína PH05 deixando inal. teradas as sequências ladeantes do cromossoma II. Seleccionou-se uma estirpe que se designou por Saccharomyces cerevisiae GRF 18 (pho-5-ΤΡΑ)♦ Exemplo 18: Melhoramento de estirpe por mutação A estirpe Saccharomyces cerevisiae GRF18 /pJDB2O7/PHO5-TPA (IA) é ainda desenvolvida para produção de TPA por mutação e selecção de baixa actividade de fosfatase ácida. Saccharomyces cerevisiae H2 4 3/pJDB 20 7/ /PH05 -TPA (IA) um mutante termo-sensível de Saccharomyces cerevisiae GRF18/pJDB2O7/PHO5 TPA (IA) ê obtido por irra_2 diação com UV (0,08 yiWcm , 15 segundos, 99,2¾ de morte). As colónias são crescidas em meio YPD osmc ticamente estabilizado contendo sorbitol 1,2M (10 g/l de Bacto Yeast Extract, 20 g/l de Bacto Peptone, 20g/l de glucose, 20 g/l de agar Bacto) e transferidas para meio YPD sem sorbitol. Saccharomyces cerevisiae H243 ê isolada como colónia de crescimento lento. Cerca de 10$ células de Saccharomyces cerevisiae H243/pJDB2O7/PHO5 -TPA (IA) são semeadas em agar YNB (6,8 1 de yeast Nitrogen Base sem aminoãcidos ; 20 g/1 de glucose, 20 g/1 de agar Bacto, suplementado com 20 mg/1 de histidina) e são irradiadas directamente com UV (0,08 -2 uWcm durante 12 segundos). A morte e de 98¾. As colonias obtidas a partir das células sobreviventes são transferidas para meio de Pi baixo e dois dias depois coradas para a actividade de fosfatase acida por um teste de sobrecamada de agar mole (48). Os mutantes negativos para a fosfatase _4 acida são obtidos com uma frequência de mutaçao de 3,2x10 . A estirpe com o título de TPA mais elevado é seleccionado e referido como Saccharomyces cerevisiae H423/pJDB2O7/PHO5-TPA (IA). As actividades de TPA de tres estirpes diferentes de levedura transformadas com pJDB2O7/PHO5-TPA (IA) estão apresentadas na Tabela 4. Tabela 4: Actividade de TPA de diferentes estirpes de Saccharomyces cerevisiae transformadas em condi ções de desrepressão (Pi baixo) Actividade de TPA (unidades/1 de cultura de células de levedura /OD^qq) GRF18/pJDB2O7/PHO5-TPA(lA) 625 H243/pJDB2O7/PHO5 -TPA(IA) 1560 H423/pJDB2O7/PHO5 -TPA(IA) 7200 • 1 Exemplo 19 : Produção de TPA por uma estirpe recombinante da levedura Saccharomyces cerevisiae numa escala de 30 1. Saccharomyces cerevisiae estirpe GRF 18/pJDB2O7/PHO5-TPA(lA) possui um plasmídeo que inclui uma marca para leucina permitindo manutenção selectiva do plasmídeo no organismo hospedeiro, um gene estrutural para o TPA humano e o promotor da fosfatase acida PH05 que permite a expressão do gene do TPA em meios com quantidades limitativas de fosfato inorgânico (condições de desrepressão). A estirpe ê mantida em culturas de agar inclinado preparadas com um meio definido sem o aminoãcido leucina para assegurar a retenção do plasmídeo. Agar recentemente inoculado e incubado durante 24 horas a 30?C. A cultura â superfície de um dos agares inclinados ê ressuspensa em 3ml de meio de pre-cultura o qual ê então transferido para o primeiro balão de pre-cultura com agitação. 0 balão de 500 ml tem uma unica saída e contem 100 ml de meio I de pre-cultura tendo o seguinte composição ( valoreq em g/1). L-asparagina, 2,0; L-histidina, 0,02 glucose, 10 ; Kt^PO^, 1,0 ; MgSO4· 7H2O, 0,5 ; NaCl 0,1 ; CaCl2~2H2O, 0,1 ; solução de vitaminas 5 jil/1 e solução de micronutriantes 5 ml/1. 0 meio e ajustado a pH7,2 usando NaOH antes da esterilização . A glucose, vitaminas e micronutrientes são esterilizados à parte e adicionados ao meio. As soluções "stock” de vitaminas e micronutrientes têm as seguintes composições (em g/1): Vitaminas - biotina, 0,0002; D-pantotenato de cãlcio , 0,04 ; acido fõlico, 0,0002; acido micotínico, 0,04 ; acido p-aminobenzõico, 0,02 ; hidrocloreto de piridoxina, 0,04; riboflãvina, 0,02 ; hidrocloreto de tiamina, 0,04; e inositol 0,2 em 1 1 de agua desionizada ; micronutrientes - acido bórico 0,05 ; CuSO "^H 0, 0,004 ; Kl, 0,01; FeCl^.óH^O, 0,02 ; MnSO^, 0,04 ; NaMoO^. 2^0, 0,02 e ZnSO^.71^0, 0,04 em 1 1 de agua desionizada. 0 meio ê preparado usando agua desionizada. A primeira pré-cultura é incubada durante 24 horas a 309C num agitador orbitol com 5cm de excentricidade a uma velocidade de 250 rev/min. Os balões com a primeira pré-cultura constituem o inoculo para os balões da segunda pré-cultura. Estes balões recebem um inoculo de 1¾ (v/v). Eles contem 100 ml de meio II de pré-cultura tendo a seguinte composição (mg/1): Extracto de levedura (Difco), 10,0 ; L-asparagina, 6,6; Κ^ΡΟ^ 1.0 ; MgSO^.7H2O, 1,0 ; L-histidina, 0,02 e D-glucose (mono-hidrato) , 33,0. 0 meio que ê preparado usando agua desionizada tem um pH de aproximadamente 6,0. A glucose é esterilizada separadamente. As condições de incubação são idênticas âs da primeira pré-cultura. 0 caldo de cultura de 3 balões ê combinado para dar um inoculo a 1¾ v/v para o fermentador principal de produção. 0 fermentador de produção tem um volume total de aproximadamente 45 1, contem 4 saliências e um agitador de turbina de disco de seis lâminas com um diâmetro de 115 mm. A velocidade de agitação é de 600 rev/min, a sobrepressão de 0,3 bar e uma velocidade de arejamento de 0,5 vol/vol/min. * ,· Η Ο fermentador contem 30 1 de um meio com as composições seguintes: (valores em g/1): L-asparagina, 2,0 ; L-histidina, 0,02 ; Kí^PO^, 0,03; MgSO^. 71^0, 0,5; NaCl, 0,1, CaCl2.2H2O, 0,1 ; KC1, ip ; D-glucose (mono -hidrato) , 20,0 ; solução de vitaminas 5 ml/1 e solução micronutrientes, 5ml/l. 0 meio ê ajustado pH 7,2 usando NaOH antes da esterilização. A glucose, vitaminas e micronutrientes são esterilizados separadamente e adicionados ao meio. As soluções "stock" de vitaminas e micronutrientes têm composição idêntica às descritas para o meio I de prê-cultura. A temperatura de fermentação ê de 309C. 0 pH desce para 5,0 onde ê controlado usando NaOH. Apos fermentação durante cerca de 20 horas atinge-se a produção máxima de TPA. A densidade óptica que atinge 2-3 unidades e a actividade de fosfatase ácida dão indicações uteis àcer ca do progresso da fermentação. 0 calor de fermentação ê arrefecido para 109C antes da colheita das células de levedura. Exemplo 20: Recuperação e purificação do TPA e pro-TPA por cromatografia com anticorpos monoclonais anti-TPA a) Preparação de extractos celulares 0 líquido de cultura (pH4) dos 30 1 de fermentação (cf.Exemplo 19) é arrefecido atê 109C e centrifugado usando uma centrífuga Alfa-Laval BRPX-207. As células separadas são ressuspensas em 21 de tampão de lise A ( NaH2P04 20mM, f^HPO^ 80mM, NaCl 150 mM, 0,01¾ de Tween, Benzamidina lOmM e EDTA lOmM). • 1 ί »» A solução é arrefecida atê 59C e passa da através de um moinho Dyno (tipo KDL-Pilot) com uma velocidade de alimentação de 101/h. 0 moinho estã equipado com 500 ml de pérolas de vidro de 0,5 -0,75 m de diâmetro e ê ligado a 3000 rev/min. Antes da adição das pérolas de vidro às células em suspensão aquelas são lavadas com 300 ml de tampão de lise A contendo 30 mg de BSA. A suspensão de células ê centrifugada ( rotor Sorvall 653, 40 min. 9000 rpm) e o sedimento rejeitado. b) Digestão do DNA^e fraccionamento com sulfato de amonio Adiciona-se 5 mg de DNAse a 2 1 da suspensão clarificada e a mistura ê agitada durante 30 min. a 49C. Subsequentemente a solução é levada a 35¾ de saturação com (NH^^SO^ (p/v) e ê agitada durante 1 hora a 49C. A solução ê centrifugada como descrito atrãs e o sedimento contendo toda a actividade de TPA ê ressuspensa em 1 1 de tampão H contendo 1¾ de Tween e 0,1¾ de SDS. A suspensão ê agitada durante pelo menos 1 hora a 49C. c) Cromatografia de imunoafinidade I) Purificação do anticorpo anti-TPA 5 ml de soro de coelho, anti -TPA (oferecido pelo Dr. E. Reich, Friedrich -Miescher-Institut Basel) são passados por uma coluna de sefarose com lisina para renovar o plasminogenio de soro. A coluna ê lavada com 1-2 volumes de PB5 pH 7,4 contendo NaCl 100 mM, KCI 2,7 mM, Na2HP04 8,lmM e KH2PO4 1,5 mM. $ rf* « « Ο líquido da coluna e de lavagens são juntos. 0 anti-soro sem plasminogênio é precipitado pela adição de (NH^)2SO^ sólido para uma concentração final de 50¾ (p/v). A solução ê mantida durante a noite a 4?C e em seguida centrifugada ( rotor Sorvall GSA,1200 rpm, 20 min.) . 0 precipitado e dissolvido num peque no volume de PB5 e dializado contra PBS contendo 0,1 ¾ de NaNj. A IgG desta solução dializada e purificada numa coluna de proteína A -sefarose (55). II) Preparação de uma coluna com anticorpo anti-TPA 13 mg de anticorpo anti-TPA purificado são dializados contra MOPS 0,1 M pH7,0. 0 anticorpo e ligado a 5 ml de Affigel 10 activado ( Biorad ) segundo as instruções do fabricante. A matrix do gel ó equilibrada com PBS contendo 1¾ de Tween 80, 0,1¾ de SDS, NaCl 0,2M, Benzamidin lOmM e 0,1¾ de NaN^. A matrix e aplicada numa coluna de 5 ml. III) Purificação de TPA por cromatografia de imunoafinidade. 0 sedimento redissolvido do fraccionamento com sulfato de amónio ê centrifugado para remover o material insolúvel ( rotor Sorvall GSA, 30 min. 12000rpm) e subsequentemente aplicado â coluna de afinidade a uma ve locidade de fluxo de cerca de lOml/hr a 49C. Depois de todo o volume ter passado através da coluna esta ê lavada com 10 volumes da coluna de PBS contendo 0,05¾. de Tween 80. A coluna ê eluída usando glicina-ΗΟΙΟ,ΙΜ pH2,5 contendo 0,1 ¾ de Tween. São colhidas fracções de 2 ml e testados para a actividade de TPA segundo o método de Rânby (49). Como substracto usa-se D-Val-Leu-Lis -pNA (Kabi -S-2251). As fracções com actividades mais elevadas são reunidas, neutralizadas usando Tris IM e concentradas por ultrafiltração usando uma membrana Amicon YM10. 0 TPA assim obtido apresenta cerca de 90¾ de pureza e estã predominantemente em forma de cadeia unica ( pro-TPA) como evidenciado por SDS-PAGE em condições de redução. 0 peso molecular aparente em condições não redutoras ê de cerca de 60 000 por SDS -PAGE (ver figura A). Figuras A e B : electroforese em gel de poliacrilamida-SDS do produto do gene TPA em levedura (Figura A) e a sua actividade enzimãtica num gel de actividade (placas com caseína) ( figura B). B:gel de actividade A:gel com SDS ad figura A (SDS-PAGE): Faixas 1 e 4 : proteínas padrão (marcadores de peso molecular) Faixas 2 e 5 : TPA de melanoma Faixas 3 e 6 : TPA de levedura Faixas 1 - 3 : em condições de redução Faixas 4 - 6 : em condições de não-redução As amostras correram num gel de 12,5¾ que se corou para proteínas usando azul Coomassie brilhante. ad. figura B (gel de actividade, cf. Exemplo 23): Faixa 1 : TPA de melanoma Faixa 2 : TPA de levedura Sobre-camada para plasminogénio de agar com caseira (referência 56) . Exemplo 21: Recuperação e purificação de TPA e pro-TPA por cromatografia de afinidade com DE-3 a. Preparação de uma coluna de DE-3 Sepharosé 26 mg do inibidor DE-3 purificado a partir de Erythrina latíssima (16) são acoplados a 5 ml de Sepharose 4b ® activada com brometo de cianogênio (Pharmacia) segundo as instruções do fabricante. A matrix ê equilibrada com tampão fosfato salino ("PBS") pH 7,4 contendo NaCl 0,4M, 0,1¾ de Triton X-100R e 0,02¾ de azida de sódio. A matrix ê então acamada numa coluna de 5 ml. b. Purificação de TPA e pro-TPA por cromatografia em DE-3 R Sepharose 4b Os extractos celulares (cf. Exemplo 20a) são tornados 0,4 M relativamente a NaCl e 0,1¾ para Triton X-lOCr^ e filtrados através de uma membrana de 0,45 ^im (Millipore). A solução ê então aplicada à coluna de DE-3 Sepharose (ver atras) a uma velocidade de fluxo de 45ml/hr â temperatura ambiente e o efluente ê rejeitado. Depois do volume total do extracto ter passado através da coluna esta ê lavada com cerca de 50 ml de PB5 contendo NaCl 0,4 M e 0,1 ¾ de Triton X-lOO*' sendo colhidos fracções de 2 ml a 49C. 0 conteúdo protêico de cada fracção ê determinado por medição da absorvância de UV a 280nm. A proteína adsorvida verificou-se ser eluída como um pico bem definido. As fracções contendo os valores mais elevados de absorvância de UV e de activida125 de fibrinolítica determinada pelo teste da I fibrina (47) são reunidas dando 8 ml de solução que se guardou a -209 C. Isto representa aproximadamente 70-80¾ da actividade total aplicada à coluna. As fracções com as actividades »· mais baixas são reunidas separadamente. A recuperação total de actividade em ambos os conjuntos de fracções monta os 90-100¾. c). Purificação de pro-TPA por cromatografia em DE-3 Sepharose 4b® na presença de um inibidor de proteíases. A cromatografia dos extractos de células de levedura ê feita de modo semelhante ao descrito no Exemplo 21b exceptuando a inclusão neste caso do inibidor de tripsina pancreãtica bãsica (BPTI) a 0,1 KlU/ml usando o teste fluorimétrico como Cbz-Gly-Gly-Arg-AMC como substracto (37) determinou-se o conteúdo em TPA e pro-TPA da solução purificada. 90¾ do TPA estã na forma de pro-enzima e 10¾ na forma activa. Assim, a conversão de pro-TPA em TPA durante o processo de purificação é inibido pelo BPTI. Exemplo 22: Separação de pro-TPA do TPA A solução de TPA e pro-TPA obtida como descrito no Exemplo 21c e ajustada a pH8,0 com Tris.HCl O,1M contendo 0,1¾ de Triton X-lOcP e tornado lmM relativamente ao diisopropilfluorofosfato. Apos incubação a 37?C durante 4 horas, a mistura ê passada através de uma coluna de 5 ml de DE-3 Sepharose® (cf. Exemplo 21a). 0 efluente contendo o TPA irreversivelmente inibido é rejeitado. A coluna e lavada com 6 volumes de coluna de PBS contendo NaCl 0,4 M e 0,1¾ de Triton X-IOC^ e subsequentemente eluída com PB5 contendo K SCN 1,6M, NaCl 0,4M e 0,1¾ de Triton X-10C^ como descrito no Exemplo 21b . As fracções apresentando as absorvân. 1 s· P0RWI « ? » ... Ή , - - - , __. «τ'. cias de UV mais elevadas são reunidas. 0 conjunto de fracções contem pro-TPA numa forma substancialmente pura uma vez que não se detecta actividade amidolítica no teste fluorimêtrico usando Cbz-Gli-Gli-Arg-AMC (37) como substrato. A actividade amidolítica assim como a fibrinolítica pode ser restabelecida por tratamento com plasmina a qual converte pro-TPA em enzima activa. Exemplo 23: Propriedades do produto do gene TPA em levedura a. Propriedades enzimãticas 0 produto do gene TPA em levedura obtido segundo o Exemplo 19 e purificado por eromatografia de afinidade em DE-3 (cf. Exemplo 21b) ê enzimãticamente activo como se mostra por clivagem de plasminogênio zimogênio em plasmina na ausência e na presença de fibrina. A actividade enzimãtica pode ser verificada pelos testes colorimêtricos descritos por Ranby (49) (cf. Exemplo 12) e por Verheijen et al. (60) , em placas de caseína e de fibrina (56) e num teste de fase solida com 12 9 I - fibrinogenio ( 47). A actividade enzimãtica em placas de caseína ê mostrada na figura B ( cf. Exemplo 20 c III). A actividade enzimãtica do TPA obtido ê grandemente estimulada pela fibrina. 0 TPA parcialmente purificado por eromatografia de afinidade em DE-3 ê testado num teste de velocidade parabólica (cf. Ranby (49)). Tomando a velocidade reacção Δ E versus Δ t^ entre e 5 minutos como uma medida da actividade enzimãtica a estimulação por concentração óptimas de fibrina ê de 10-20 vezes ( ca. 100 yig /ml) . 0 efeito pode ser observado com fragmentos de fibrina obtidos por digestão com CnBr |
EP0164566A3 (de) * | 1984-05-11 | 1986-10-29 | LGA Gastechnik GmbH | Sicherheitseinrichtung für Flüssiggastanks |
EP0480480A3 (en) * | 1984-05-11 | 1992-06-10 | Chiron Corporation | Enhanced yeast transcription employing hybrid promoter region constructs |
US4895800A (en) * | 1985-11-26 | 1990-01-23 | Phillips Petroleum Company | Yeast production of hepatitis B surface antigen |
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1985
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