KR20240021866A - 효소적 핵산 합성을 위한 조성물 및 방법 - Google Patents
효소적 핵산 합성을 위한 조성물 및 방법 Download PDFInfo
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Abstract
본 개시내용은 핵산의 주형 독립적 효소 합성에 유용한 조성물 및 방법을 기술한다.
Description
<정부 라이센스 권리>
본 발명은 미국국립보건원(National Institutes of Health)에서 부여한 Award Number1R43HG010995-01A1 및 Unique Federal Award Identification Number (FAIN) R43HG010995 하에 정부 지원으로 이루어졌다. 상기 정부는 발명에 대한 특정 권리를 갖는다.
<서열 목록의 통합>
크기가 약 173KB인 (PG0020_sequence_listing_revised_10-28-21_ST25.txt) 이름의 ASCII 텍스트 파일로 전자적으로 제출된 서열 목록의 내용은 2021년 10월 28일에 작성되었으며 2022년 6월 13일에 ePCT를 통해 전자적으로 제출되었다.
합성 DNA 및 RNA를 생산하는 현재 방법인 화학적 올리고뉴클레오타이드 합성(COS, Chemical oligonucleotide synthesis)은 거의 40년이 되었으며 기능 유전체학, 합성 생물학, DNA 기반 데이터 저장, 빠르고 저렴한 DNA 합성에 의존하는 의료 응용 분야 등의 분야에서 새로운 발견을 하는데 제한이 되었다. COS 비용은 지난 분기 동안 20배만 향상되었으며(예를 들어 Bioeconomy Capital 웹 사이트의 바이오경제 대시보드에 표시되는 데이터 참조) 합성 DNA에 대한 수요 증가를 따라잡지 못했다. 또한 COS는 최대 200개 정도의 뉴클레오타이드를 갖는 핵산 가닥으로 제한되며 정교한 장비와 생산 프로세스를 사용하는 대규모 중앙 집중식 시설이 필요하다. 합성 핵산에 대한 수요가 급격히 증가함에 따라 긴 핵산 분자를 전달할 수 있는 새롭고 신속하며 저렴한 합성 경로가 필요하다. 자연에는 DNA와 RNA 중합효소가 풍부하기 때문에 효소적 핵산 합성 경로가 많은 주목을 받고 있다.
효소 올리고뉴클레오타이드 합성(EOS)은 최근 흥미로운 발견과 발전(Palluk 2018, Perkel 2019, Hoff 2020, Lee 2020)을 통해 수년 동안 다양한 상업 그룹에 의해 추구되어 왔다 (Efcavitch 2016, Hiatt 1995, Hiatt 1995a).
대부분의 EOS 전략은 시험관 내에서 단일 가닥 DNA의 3' 말단에 뉴클레오타이드를 추가할 수 있는 TIDP(주형 독립적 DNA 중합효소, template-independent DNA polymerases)인 TdT(말단 데옥시뉴클레오티딜 전이효소, terminal deoxynucleotidyl transferases)를 사용한다(Deibel 1980, Fowler 2006, Motea 2010, Jensen 2018, Loc'h 2018, Deshpande 2019, Sarac 2019). 알려진 TdT는 높은 가공성 또는 효소의 높은 온-오프 속도를 통해(Gouge 2013) 수백 개의 뉴클레오타이드 길이의 DNA를 중합한다(Deibel 1980, Delarue 2002, Fowler 2006, Motea 2010, Jensen 2018, Loc'h 2018, Sarac 2019). 비-TdT 효소의 TIDP 활성은 광범위하게 연구되지 않았지만, 다른 DNA 중합효소, 특히 DNA 복구 과정에 관여하는 DNA 중합효소도 시험관 내에서 주형 독립적 DNA 중합효소(TIDP) 활성을 갖는 것으로 나타났다(Clark 1988, Domnguez 2000, Ruiz 2001, Juarez 2006, Moon 2007, Moon 2007a, Hogg 2012, Moon 2014, Kent 2016, Frank 2017, Yang 2018, Chang 2019).
정의된 길이와 서열의 폴리뉴클레오타이드를 생성하기 위해 현재 EOS 프로세스는 3' 차단 뉴클레오타이드를 사용하며 각 추가 주기 후에 차단 그룹을 제거한다(도 1A). 3' 차단 그룹(3' blocking group)은 추가 주기당 여러 뉴클레오타이드의 추가를 방지한다.
그러나 3'-차단된 뉴클레오타이드에는 이 분야의 발전을 제한하는 여러 가지 단점이 있다. 첫째, 대부분의 천연 DNA 중합효소는 3' 변형이 있는 뉴클레오타이드를 매우 비효율적으로 통합하며 또한 뚜렷한 염기 선호도와 서열 특이성을 나타낸다. 둘째, 3' 차단 그룹의 화학적 특성은 첨가 단계 동안 자발적 또는 효소 촉매에 의한 제거를 방지할 수 있을 만큼 충분히 안정적이어야 하고 다음 첨가 단계를 준비하기 위해 완전히 제거 가능해야 하기 때문에 매우 중요하다. 이 균형은 달성하기 어렵고 바람직한 품질을 가진 소수의 차단 그룹 화학 분야로 제한되었다. 셋째, 효소는 뉴클레오타이드 화학과 효소 최적화의 상호 연결된 문제를 일으키는 3' 차단 그룹을 수용해야 한다. 넷째, 이 전략의 차단 해제 단계는 효소 합성 공정에 화학 반응 단계를 추가하여 공정 복잡성을 증가시키고 잠재적으로 값비싸고 독성이 있는 화학 물질의 사용을 수반한다.
천연 또는 차단되지 않은 뉴클레오사이드 트리포스페이트를 사용하는 올리고뉴클레오타이드 합성에 대한 대안적인 접근 방식이 설명되었다(Schott 1984). 주형 독립적 핵산 중합효소에 의한 다중 뉴클레오타이드의 프로세스적 추가로 인해, 이 방법은 각 추가 주기(addition cycle) 후에 단일 뉴클레오타이드 추가를 받은 올리고뉴클레오타이드 분자가 0, 2개 또는 그 이상의 뉴클레오타이드를 받은 올리고뉴클레오타이드로부터 분리되어야 함을 요구한다. 각 추가 주기 이후 올리고뉴클레오타이드 정제에 대한 요구 사항으로 인해 이 방법의 유용성이 제한되었다.
효소 올리고뉴클레오타이드 합성 문제를 단순화하고 효율적인 효소 올리고뉴클레오타이드 합성 공정을 위한 차별화된 접근 방식을 만들기 위해, 우리는 천연 뉴클레오타이드만을 사용하는 도 1B에 표시된 전략을 개발했다. 뉴클레오타이드를 효율적으로 추가한 다음 전위(translocate)에 실패하고 DNA 주형과 연결된 상태를 유지하는 TIDP는 합성 주기당 단일 뉴클레오타이드만 안정적으로 추가한다. 이로써 효소는 올리고뉴클레오타이드 기질의 3' 말단에 하나 이상의 뉴클레오타이드가 추가되는 것을 방지하고 변형된 뉴클레오타이드의 필요성을 제거한다. 새로운 주기(cycle)를 시작하기 전에 뉴클레오타이드를 제거하고 세척, 가열 및/또는 카오트로픽 염(chaotropic salt)을 사용하여 효소를 분리한다. 이 프로세스에 적합한 TIDP의 진화가 크게 간소화되고 DNA 합성 비용이 크게 절감된다. Primordial Genetics의 비용 모델은 이러한 EOS 프로세스가 소규모(fmol) 및 중간(nmol-μmol) 합성 규모에서 COS에 비해 10배~100배의 비용 이점을 갖는다는 것을 보여준다.
본 개시내용은 단일 가닥 올리고뉴클레오타이드의 말단에 단일 뉴클레오타이드를 통합하는 능력을 갖는 1세대 DNA 합성 효소 세트를 사용하는 이러한 독특한 DNA 합성 접근법에 대한 타당성을 입증한다.
합성 DNA에 대한 응용이 빠르게 성장함에 따라 이 분야의 상업적 기회는 엄청나다. 전 세계 올리고뉴클레오타이드 합성 시장 규모는 2018년 43억 달러였으며 연평균 성장률(CAGR) 10-12.5%로 성장하여 2025년까지 80억 달러 이상에 이를 것으로 예상된다 (Global Oligonucleotide Synthesis Market Size 2018). 합성 DNA의 주요 응용 분야에는 분자 및 합성 생물학 R&D, 유전체학(표적 강화), 치료제, 진단(DNA 마이크로어레이, PCR 및 FISH), CRISPR/Cas9 시스템, 나노기술 및 DNA 기반 데이터 저장 및 DNA 컴퓨팅과 같은 신기술이 포함된다 (Global Oligonucleotide Synthesis Market Size 2018, Lee 2018, Jensen 2018, Lee 2019)
본 개시내용은 기질로서 자유(free) 또는 차단되지 않은 3' 하이드록실기(unblocked 3' hydroxyl group)를 갖는 뉴클레오사이드 트리포스페이트(이하 '차단되지 않은 뉴클레오사이드 트리포스페이트(unblocked nucleoside triphosphate)'라고 함)를 사용하여 올리고뉴클레오타이드 합성을 위한 신규한 효소적 경로를 설명한다. 지금까지 기술된 TIDP 활성을 갖는 DNA 중합효소는 일반적으로 시험관 내에서 트리포스페이트와 반응할 때 단일 가닥 올리고뉴클레오타이드 또는 폴리뉴클레오타이드 말단에 뉴클레오타이드가 순차적으로 첨가되는 것을 보여준다. 본 개시내용은 차단되지 않은 뉴클레오사이드 트리포스페이트와 함께 사용될 때 올리고뉴클레오타이드의 3' 말단에 단일 뉴클레오타이드를 추가하는 능력을 갖는 DNA 중합효소를 설명한다.
본 개시내용은 공지된 DNA 중합효소 메커니즘에 확고히 뿌리를 두고 있다. 간단히 말해서, 모든 DNA 중합효소는 6가지 주요 기계적 단계를 거치는 것으로 알려져 있다(Berdis 2009, Beard 2014, Berdis 2014): 1) DNA 기질에 결합하는 중합효소; 2) 뉴클레오사이드 트리포스페이트와 초기 삼원 복합체(initial ternary complex)의 형성; 3) 생산적인 삼원 기질 복합체(ternary substrate complex)로 이어지는 형태 변화; 4) 화학 후 생성물 삼원 복합체(product ternary complex)로 이어지는 촉매작용; 5) 생성물(PPi) 출시로 이어지는 구조적 변화, 그리고 6) DNA 기질로부터 다음 단계의 뉴클레오타이드 첨가 또는 중합효소 해리를 준비하기 위한 중합효소 전위. 이러한 다양한 기계적 단계는 중합효소의 다양한 도메인에 의해 매개된다(Kaminsky 2020).
중합효소 전위(Polymerase translocation)는 특정 DNA 중합효소 서열 및 도메인과 연관되어 있는 것으로 알려져 있으며(Samkurashvili 1996, Rechkoblit 2006, Golosov 2010, Dahl 2014, Ren 2016, Yang 2018, Hoitsma 2020), 기질로부터 해리 속도가 크게 다른 중합효소가 보고되었다(Andrade 2009, Zahn 2011). 전위 속도에 영향을 미치는 DNA 및 RNA 중합효소 모두에서 돌연변이가 확인되었으며(Samkurashvili 1996, Dahl 2014, Ren 2016), 중합효소 전위는 DNA 및 RNA 중합효소에서 발견되는 특정 도메인 및 서열 모티프와 연관되어 있다(Samkurashvili 1996, Rechkoblit 2006, Golosov 2010, Dahl 2014, Hoitsma 2020). 따라서 차단되지 않은 단일 뉴클레오타이드를 추가하고 전위가 불가능하여 다른 뉴클레오타이드를 추가하지 못하는 핵산 중합효소를 개발하는 것이 가능하다.
핵산 중합효소는 다른 종류로 분류되며, 한 종류 내의 중합효소는 다른 종류에 있는 중합효소와 구별되는 특정 서열이나 특성을 나타낸다. 예를 들어, DNA 중합효소는 A, B, C, D, X, Y 및 RT 계열로 분류된다(Bebenek 2002, Ramadan 2004, Jarosz 2007, Guo 2009, Uchiyama 2009, Yamtich 2010, Berdis 2014, Maxwell 2014, Moon 2014, Trakselis 2014, Yang 2014, Vaisman 2017, Yang 2018, Hoitsma 2020, Kazlauskas 2020). 서로 다른 계열의 중합효소는 핵산 복제, 복구 및 재조합에서 서로 다른 생물학적 기능을 가지고 있다. 다양한 계열의 정제된 중합효소는 위에 나열된 참고문헌에 예시된 것처럼 종종 서로 다른 시험관 내 활성 세트를 나타낸다.
핵산 중합효소는 또한 핵산 중합에서 특정 서열에 대한 강한 서열 특이성 또는 선호도를 나타내는 것으로 알려져 있다. 핵산 중합효소는 또한 핵산을 중합할 때 염기 특이성을 나타내는 것으로 나타났다(Fiala 2007, Hoitsma 2020).
DNA 중합효소의 알려진 특성을 기반으로, 다음을 포함하되 이에 국한되지 않는 여러 뉴클레오타이드의 프로세스적 추가 위험 없이 단일 가닥 핵산 분자의 3' 말단에 단일 뉴클레오타이드를 추가할 수 있는 다양한 잠재적인 방법이 있다: 1) 변형된 핵산 분자의 3' 말단 서열에 대해 서열 특이성이 높은 중합효소의 사용; 이 말단 서열 특이성은 특정 유형의 뉴클레오타이드(예를 들어 A, C, G, T, U 또는 I)를 통합하는 중합효소의 선호 측면에서 염기 특이성과 연결될 수도 있고 연결되지 않을 수도 있다; 2) 뉴클레오타이드 추가(위의 6단계) 후에 전위할 수 없고 뉴클레오타이드 추가 후에도 핵산 분자의 3' 말단과 결합된 채로 남아 있는 DNA 중합효소의 사용; 3) 이들의 조합; 그리고 4) TIDP가 핵산 기질에서 비처리적으로(non-processively) 작용하고 주형에 독립적인 방식으로 차단되지 않은 단일 뉴클레오타이드만 추가할 수 있도록 하는 기타 메커니즘.
본 개시내용은 뉴클레오사이드 트리포스페이트 단량체(nucleoside triphosphate monomer) 상의 3' 차단기(3' blocking group)를 사용하지 않고 주형-독립적 핵산 중합효소(TINAP, template-independent nucleic acid polymerase)에 의해 핵산 기질(nucleic acid substrate)에 단일 뉴클레오타이드를 첨가하는 것을 수반하는 핵산의 효소적 드노보 합성(de novo synthesis)에 대한 새로운 접근 방식을 설명한다. 본 개시내용은 또한 주형에 독립적인 방식으로 핵산의 3' 말단에 단일 뉴클레오타이드를 추가할 수 있는 효소를 설명한다. 이 놀라운 발견은 DNA 중합효소가 알려져 있고 작동한다고 생각되는 진보적인 방식과 모순된다. 결과적으로, 이러한 효소 또는 이의 변형된 유도체는 한 번에 하나의 뉴클레오타이드씩 핵산의 3' 말단에 뉴클레오타이드를 제어하여 추가해야 하는 EOS 공정의 개발에서 유용성을 찾는다. 본 개시내용은 산업, 의료, 진단, 농업 및/또는 R&D 용도를 위한 핵산 합성에 사용되는 공정에서 이러한 효소의 사용을 설명한다.
도 1A. 올리고뉴클레오타이드에 3'-차단된 뉴클레오타이드를 주기적으로 첨가하여 효소적 올리고뉴클레오타이드 합성(enzymatic oligonucleotide synthesis)을 도식적으로 표현 (Jensen 2018 참조). 비드에 결합된 올리고뉴클레오타이드(왼쪽 상단)는 3'-차단된 뉴클레오사이드 트리포스페이트(상단) 및 비드에 뉴클레오타이드 추가를 촉매하는 효소(오른쪽 상단)와 결합된다. 효소와 과도한 뉴클레오사이드 트리포스페이트(표시되지 않음)을 제거한 후, 3' 보호 그룹이 절단되어(하단) 또 다른 추가의 기질인 자유 3' 말단이 남는다. 합성이 완료되면 보호가 해제된 올리고뉴클레오타이드가 비드에서 절단될 수 있다(왼쪽 하단). 다이어그램은 DNA 올리고에 C 잔기의 추가를 보여주지만 임의의 RNA 또는 DNA 올리고뉴클레오타이드, 또는 이의 변형된 형태 또는 키메라에 추가된 임의의 뉴클레오타이드에 동일하게 적용된다.
도 1B. 올리고뉴클레오타이드에 뉴클레오타이드를 주기적으로 첨가하여 효소적 올리고뉴클레오타이드 합성을 도식적으로 표현하여 보호기 제거가 어떻게 핵산 합성 주기를 단순화할 수 있는지 보여준다.
도 1C. 차단되지 않은 뉴클레오타이드를 올리고뉴클레오타이드에 주기적으로 첨가함으로써 효소적 올리고뉴클레오타이드 합성을 도식적으로 표현. 비드에 결합된 올리고뉴클레오타이드(왼쪽 상단)는 자유 3' 말단(상단)이 있는 뉴클레오사이드 트리포스페이트와 단일 뉴클레오타이드를 비드에 추가하는 것을 촉매하는 효소(오른쪽 상단)와 결합된다. 상기 효소(왼쪽 아래)와 과도한 뉴클레오사이드 트리포스페이트(표시되지 않음)을 제거한 후 주기가 반복될 수 있다. 합성이 완료되면 올리고뉴클레오타이드가 비드에서 절단될 수 있다(왼쪽 하단). 다이어그램은 DNA 올리고에 C 잔기의 추가를 보여주지만 임의의 RNA 또는 DNA 올리고뉴클레오타이드, 또는 이의 변형된 형태 또는 키메라에 추가된 임의의 뉴클레오타이드에 동일하게 적용된다.
도 1D. 차단되지 않은 뉴클레오타이드를 올리고뉴클레오타이드에 주기적으로 추가함으로써 효소적 올리고뉴클레오타이드 합성을 개략적으로 표현, 추가 주기마다 단일 뉴클레오타이드가 추가되는 하나의 가능한 메커니즘을 보여줌. 비드에 결합된 올리고뉴클레오타이드(왼쪽 상단)는 자유 3' 말단(상단)이 있는 뉴클레오사이드 트리포스페이트와 단일 뉴클레오타이드를 비드에 추가하는 것을 촉매하는 효소(오른쪽 상단)와 결합된다. 뉴클레오타이드를 첨가한 후에도 효소는 올리고뉴클레오타이드의 3' 말단에 결합된 상태로 남아 있어 추가적인 핵산 중합을 방지한다. 효소(왼쪽 아래)와 과도한 뉴클레오사이드 트리포스페이트(표시되지 않음)을 제거한 후 주기가 반복될 수 있다. 합성이 완료되면 올리고뉴클레오타이드가 비드에서 절단될 수 있다(왼쪽 하단). 다이어그램은 DNA 올리고에 C 잔기의 추가를 보여주지만 임의의 RNA 또는 DNA 올리고뉴클레오타이드, 또는 이의 변형된 형태 또는 키메라에 추가된 임의의 뉴클레오타이드에 동일하게 적용된다.
도 2: 혼합된 뉴클레오사이드 트리포스페이트(dATP, dCTP, dGTP 및 dTTP의 등몰 혼합물)와 올리고뉴클레오타이드 기질(서열 번호: 42-45)의 혼합을 포함하는 뉴클레오타이드 첨가 반응의 결과. 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. 본 개시내용에 나열된 모든 효소에 사용되는 식별자인 테스트된 효소의 EDS 번호(자세한 내용은 표 1 참조)가 겔 이미지 아래에 표시되어 있다. 테스트된 효소는 기질에 다양한 길이의 서열이 추가되는 것을 보여준다.
도 3: 서로 다른 염기로 끝나는 올리고뉴클레오타이드 기질에 단일 뉴클레오타이드를 제어하여 추가한 결과. A. 반응 후 겔에 의해 분석되는 다양한 올리고뉴클레오타이드 기질에 단일 뉴클레오타이드의 첨가. 단일 가닥 DNA 사다리는 가장 왼쪽 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. B. 첫 번째 첨가 단계 후 올리고뉴클레오타이드를 정제하여 올리고뉴클레오타이드 기질에 두 개의 뉴클레오타이드를 순차적으로 첨가한다. 단일 가닥 DNA 사다리는 레인 1의 왼쪽과 레인 6의 왼쪽에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. 아래 표의 "3' 말단 염기" 열에는 각 레인에 존재하는 주요 올리고뉴클레오타이드의 3' 말단 염기가 나열되어 있다.
도 4: Oligo Pro II 모세관 전기영동 기기(Agilent Technologies, Santa Clara, CA)에서 수행된 효소적 뉴클레오타이드 첨가 전후의 올리고뉴클레오타이드의 대표적인 모세관 전기영동 분리 크로마토그램. 크로마토그램에 표시된 모든 반응은 dTTP 및 Oligo: PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)을 사용했다. 각 샘플에 존재하는 올리고뉴클레오타이드의 길이를 명확하게 지정하기 위해 올리고뉴클레오타이드 표준이 있거나 없는 샘플에 대한 이중 분석을 수행했다. 사용된 올리고뉴클레오타이드 표준은 PG1350 (GCGTCACGCTACCAACCA, 서열 번호 41); PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5870 (GTCCTCAATCGCACTGGAAACATCAAGGTC, 서열 번호 51); and PG5871 (GTCCTCAATCGCACTGGAAACATCAAGGTCATACGGAACG, 서열 번호 52)이다: 미반응(즉, 효소 없음) 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). B: 올리고뉴클레오타이드 표준과 결합된 미반응(즉, 효소 없음) 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). C: dTTP 및 효소 EDS082와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). D: dTTP 및 효소 EDS082와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후에 결합되었다. E: dTTP 및 효소 EDS054와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). F: dTTP 및 효소 EDS054와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후에 결합되었다. G: dTTP 및 효소 EDS066과 반응된 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). H: dTTP 및 효소 EDS066과 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후 결합되었다.
도 5: 다양한 길이의 서열을 기질에 첨가하는 것을 보여주는 뉴클레오타이드 첨가 반응의 결과.A: ATP, CTP, GTP 및 UTP와 효소 EDS015, EDS017, EDS029, EDS048, EDS053, EDS054 또는 EDS066의 등몰 혼합물을 갖는 올리고뉴클레오타이드 기질(서열 번호: 42-45). 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 겔 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. B: ATP, CTP, GTP 및 UTP와 효소 EDS017, EDS024, EDS029, EDS030, EDS053, EDS054, EDS066 또는 EDS082의 등몰 혼합물을 갖는 단일 올리고뉴클레오타이드 기질(서열 번호 45). 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다.
도 1B. 올리고뉴클레오타이드에 뉴클레오타이드를 주기적으로 첨가하여 효소적 올리고뉴클레오타이드 합성을 도식적으로 표현하여 보호기 제거가 어떻게 핵산 합성 주기를 단순화할 수 있는지 보여준다.
도 1C. 차단되지 않은 뉴클레오타이드를 올리고뉴클레오타이드에 주기적으로 첨가함으로써 효소적 올리고뉴클레오타이드 합성을 도식적으로 표현. 비드에 결합된 올리고뉴클레오타이드(왼쪽 상단)는 자유 3' 말단(상단)이 있는 뉴클레오사이드 트리포스페이트와 단일 뉴클레오타이드를 비드에 추가하는 것을 촉매하는 효소(오른쪽 상단)와 결합된다. 상기 효소(왼쪽 아래)와 과도한 뉴클레오사이드 트리포스페이트(표시되지 않음)을 제거한 후 주기가 반복될 수 있다. 합성이 완료되면 올리고뉴클레오타이드가 비드에서 절단될 수 있다(왼쪽 하단). 다이어그램은 DNA 올리고에 C 잔기의 추가를 보여주지만 임의의 RNA 또는 DNA 올리고뉴클레오타이드, 또는 이의 변형된 형태 또는 키메라에 추가된 임의의 뉴클레오타이드에 동일하게 적용된다.
도 1D. 차단되지 않은 뉴클레오타이드를 올리고뉴클레오타이드에 주기적으로 추가함으로써 효소적 올리고뉴클레오타이드 합성을 개략적으로 표현, 추가 주기마다 단일 뉴클레오타이드가 추가되는 하나의 가능한 메커니즘을 보여줌. 비드에 결합된 올리고뉴클레오타이드(왼쪽 상단)는 자유 3' 말단(상단)이 있는 뉴클레오사이드 트리포스페이트와 단일 뉴클레오타이드를 비드에 추가하는 것을 촉매하는 효소(오른쪽 상단)와 결합된다. 뉴클레오타이드를 첨가한 후에도 효소는 올리고뉴클레오타이드의 3' 말단에 결합된 상태로 남아 있어 추가적인 핵산 중합을 방지한다. 효소(왼쪽 아래)와 과도한 뉴클레오사이드 트리포스페이트(표시되지 않음)을 제거한 후 주기가 반복될 수 있다. 합성이 완료되면 올리고뉴클레오타이드가 비드에서 절단될 수 있다(왼쪽 하단). 다이어그램은 DNA 올리고에 C 잔기의 추가를 보여주지만 임의의 RNA 또는 DNA 올리고뉴클레오타이드, 또는 이의 변형된 형태 또는 키메라에 추가된 임의의 뉴클레오타이드에 동일하게 적용된다.
도 2: 혼합된 뉴클레오사이드 트리포스페이트(dATP, dCTP, dGTP 및 dTTP의 등몰 혼합물)와 올리고뉴클레오타이드 기질(서열 번호: 42-45)의 혼합을 포함하는 뉴클레오타이드 첨가 반응의 결과. 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. 본 개시내용에 나열된 모든 효소에 사용되는 식별자인 테스트된 효소의 EDS 번호(자세한 내용은 표 1 참조)가 겔 이미지 아래에 표시되어 있다. 테스트된 효소는 기질에 다양한 길이의 서열이 추가되는 것을 보여준다.
도 3: 서로 다른 염기로 끝나는 올리고뉴클레오타이드 기질에 단일 뉴클레오타이드를 제어하여 추가한 결과. A. 반응 후 겔에 의해 분석되는 다양한 올리고뉴클레오타이드 기질에 단일 뉴클레오타이드의 첨가. 단일 가닥 DNA 사다리는 가장 왼쪽 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. B. 첫 번째 첨가 단계 후 올리고뉴클레오타이드를 정제하여 올리고뉴클레오타이드 기질에 두 개의 뉴클레오타이드를 순차적으로 첨가한다. 단일 가닥 DNA 사다리는 레인 1의 왼쪽과 레인 6의 왼쪽에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. 아래 표의 "3' 말단 염기" 열에는 각 레인에 존재하는 주요 올리고뉴클레오타이드의 3' 말단 염기가 나열되어 있다.
도 4: Oligo Pro II 모세관 전기영동 기기(Agilent Technologies, Santa Clara, CA)에서 수행된 효소적 뉴클레오타이드 첨가 전후의 올리고뉴클레오타이드의 대표적인 모세관 전기영동 분리 크로마토그램. 크로마토그램에 표시된 모든 반응은 dTTP 및 Oligo: PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)을 사용했다. 각 샘플에 존재하는 올리고뉴클레오타이드의 길이를 명확하게 지정하기 위해 올리고뉴클레오타이드 표준이 있거나 없는 샘플에 대한 이중 분석을 수행했다. 사용된 올리고뉴클레오타이드 표준은 PG1350 (GCGTCACGCTACCAACCA, 서열 번호 41); PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5870 (GTCCTCAATCGCACTGGAAACATCAAGGTC, 서열 번호 51); and PG5871 (GTCCTCAATCGCACTGGAAACATCAAGGTCATACGGAACG, 서열 번호 52)이다: 미반응(즉, 효소 없음) 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). B: 올리고뉴클레오타이드 표준과 결합된 미반응(즉, 효소 없음) 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). C: dTTP 및 효소 EDS082와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). D: dTTP 및 효소 EDS082와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후에 결합되었다. E: dTTP 및 효소 EDS054와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). F: dTTP 및 효소 EDS054와 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후에 결합되었다. G: dTTP 및 효소 EDS066과 반응된 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45). H: dTTP 및 효소 EDS066과 반응한 올리고뉴클레오타이드 PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45)은 올리고뉴클레오타이드 표준과의 반응 후 결합되었다.
도 5: 다양한 길이의 서열을 기질에 첨가하는 것을 보여주는 뉴클레오타이드 첨가 반응의 결과.A: ATP, CTP, GTP 및 UTP와 효소 EDS015, EDS017, EDS029, EDS048, EDS053, EDS054 또는 EDS066의 등몰 혼합물을 갖는 올리고뉴클레오타이드 기질(서열 번호: 42-45). 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 겔 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다. B: ATP, CTP, GTP 및 UTP와 효소 EDS017, EDS024, EDS029, EDS030, EDS053, EDS054, EDS066 또는 EDS082의 등몰 혼합물을 갖는 단일 올리고뉴클레오타이드 기질(서열 번호 45). 단일 가닥 DNA 사다리는 "M" 레인에 표시되며, 젤 이미지 왼쪽의 라벨에 표시된 분자 크기를 포함한다.
명세서 및 청구범위의 해석을 위해 다음 약어 및 정의가 사용될 것이다.
본원에 사용된 용어 "포함한다(comprises, includes)", "포함하는(comprising, including)", "가진다", "갖는", "함유한다", "함유하는", "~을 특징으로 하는" 또는 이들의 임의의 기타 변형 용어는 비배타적 포함을 망라하고자 하는 것이다. 예를 들어, 요소들의 목록을 포함하는 조성물, 혼합물, 공정, 방법, 물품 또는 장치는 반드시 그러한 요소만으로 한정되는 것이 아니라, 명시적으로 열거되지 않은 다른 요소들 또는 그러한 조성물, 혼합물, 공정, 방법, 물품 또는 장치에 고유한 다른 요소들을 포함할 수도 있다.
첨가 주기(Addition cycle): 본 명세서에 사용된 바와 같이, 이 문구는 두 번 이상의 추가 라운드를 포함하는 핵산 합성 과정에서 한 라운드의 뉴클레오타이드 추가를 의미한다. 각 첨가 사이클에서, 합성되는 단일 가닥 핵산은 뉴클레오사이드 트리포스페이트 및 핵산 중합효소와 결합되고 핵산 중합효소가 활성화되는 반응 조건 하에서 배양되어 단일 가닥 핵산에 뉴클레오타이드가 추가된다.
핵산 중합효소의 염기 특이성: 이 문구는 다른 염기와 비교하여 특정 염기를 포함하는 뉴클레오타이드를 추가하는 핵산 중합효소의 선호를 나타낸다. 예를 들어, dTTP를 선호하는 DNA 중합효소는 A, C 또는 G와 같은 다른 염기를 포함하는 뉴클레오타이드보다 dTMP(deoxythymidine monophosphate) 잔기를 핵산의 3' 말단에 더 효율적으로 추가한다. 또 다른 예에서, 등몰량의 뉴클레오사이드 트리포스페이트 dATP, dCTP, dGTP 및 dTTP를 포함하는 혼합 반응에서, dTTP를 선호하는 DNA 중합효소는 다른 세 가지 염기 A, C 또는 G를 포함하는 뉴클레오타이드보다 핵산의 3' 말단에 더 많은 수의 dTMP 잔기를 추가한다.
키메라 핵산: 본 명세서에 사용된 바와 같이, 키메라 핵산은 리보뉴클레오타이드와 데옥시리보뉴클레오타이드 잔기의 혼합물을 함유하는 핵산 분자를 의미한다. 혼합물은 임의의 수의 리보뉴클레오타이드 잔기가 임의의 수의 데옥시뉴클레오타이드 잔기와 함께 동일한 핵산 가닥에 존재함을 의미한다.
상보적인 뉴클레오타이드 서열: 본 명세서에 사용된 바와 같이, 상보적 뉴클레오타이드 서열은 모든 염기가 5'에서 3' 극성이 반대인 다른 폴리뉴클레오타이드 서열과 염기쌍을 형성할 수 있는 폴리뉴클레오타이드 서열이고, 각 폴리뉴클레오타이드 사슬의 모든 염기는 대응물과 쌍을 이루어 염기쌍을 형성한다.
제어 요소: '제어 요소'라는 용어는 코딩 서열의 업스트림(5' 비코딩 서열), 내부 또는 다운스트림(3' 비코딩 서열)에 위치하고 전사, RNA 프로세싱 또는 안정성, 또는 연관된 코딩 서열의 번역에 영향을 미치는 뉴클레오타이드 서열을 지칭한다. 조절 서열(Regulatory sequence)에는 프로모터, 번역 리더 서열(translation leader sequences), 인트론, 폴리아데닐화 인식 서열(polyadenylation recognition sequences), RNA 프로세싱 부위, 효과기 결합 부위(effector binding sites) 및 스템-루프 구조(stem-loop structure)가 포함되지만 이에 국한되지는 않는다.
축퇴 서열: 이 출원에서, 축퇴 서열은 특정 서열 위치가 집단 내의 서로 다른 분자 또는 클론 간에 다른 서열 집단으로 정의된다. 서열 차이는 단일 뉴클레오타이드 또는 임의 개수의 다중 뉴클레오타이드일 수 있으며, 예를 들면 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000개의 뉴클레오타이드 또는 그 사이의 임의의 수 이다. 축퇴 서열의 서열 차이는 서열, 분자 또는 클론 집단 내의 해당 위치에 2, 3 또는 4개의 서로 다른 뉴클레오타이드가 존재함을 의미할 수 있다. 서열의 특정 위치에 있는 축퇴 뉴클레오타이드의 예는 A 또는 C; A 또는 G; A 또는 T; C 또는 G; C 또는 T; G 또는 T; A, C 또는 G; A, C 또는 T; A, G 또는 T; C, G 또는 T; A, C, G 또는 T.
DNA: DNA는 디옥시리보뉴클레오타이드의 중합체인 핵산이다. DNA는 단일 가닥 또는 이중 가닥 형태로 발생한다. 본 명세서에 사용된 바와 같이, DNA는 각각 CH2 형태의 2' 탄소를 갖는 뉴클레오타이드 잔기를 함유한다.
효소적 올리고뉴클레오타이드 합성(Enzymatic oligonucleotide synthesis, EOS): 본 명세서에 사용된 바와 같이, 이는 핵산의 말단에 단일 뉴클레오타이드를 단계적으로 효소적으로 첨가하여 한 번에 하나의 뉴클레오타이드씩 새로운 핵산을 생성함으로써 핵산을 합성하는 제어된 효소 과정이다.
발현: 본원에 사용된 용어 "발현(expression)"은 개시된 핵산으로부터 유래된 센스(mRNA) 또는 안티센스 RNA의 전사 및 안정적인 축적뿐만 아니라 mRNA의 번역 산물로서 폴리펩티드의 축적을 의미한다.
유리 뉴클레오타이드(Free nucleotide): 본원에 사용된 바와 같이, 일반적으로 용액 상태의 단량체성 뉴클레오타이드를 의미한다.
전장 오픈 리딩 프레임(Full-length Open Reading Frame): 본 명세서에 사용된 바와 같이, 전장 오픈 리딩 프레임은 세포 또는 유기체에서 발현되는 바와 같이 천연 개시 코돈부터 천연 최종 아미노산 코딩 코돈까지 확장되는 전장 단백질을 코딩하는 오픈 리딩 프레임을 의미한다. 특정 오픈 리딩 프레임 서열이 세포나 유기체 내에서 발현되는 여러 개의 별개의 전장 단백질을 생성하는 경우, 여러 개의 서로 다른 단백질 중 하나를 인코딩하는 이 서열 내의 각 오픈 리딩 프레임은 전체 길이로 간주된다. 전장 오픈 리딩 프레임 은 연속적이거나 인트론에 의해 중단될 수 있다.
전장 단백질(Full-length Protein): 본 명세서에 사용된 바와 같이, 전장 단백질은 세포 또는 유기체의 게놈에서 암호화되고 세포 또는 유기체에서 발현되는 천연 첫 번째 아미노산에서 천연 최종 아미노산까지 확장되는 폴리펩티드이다.
유전자: "유전자"라는 용어는 선택적으로 코딩 서열 앞(5' 비코딩 서열) 및 뒤(3' 비코딩 서열)의 조절 서열을 포함하는, 특정 단백질로서 발현될 수 있는 핵산 단편을 의미한다. "네이티브 유전자"는 자연 숙주 유기체에서 자연적으로 발견되는 유전자를 의미한다. "내추럴 유전자"는 프로모터 및 터미네이터와 같은 천연 제어 서열을 갖춘 완전한 유전자를 의미한다. "키메라 유전자"는 자연에서 함께 발견되지 않는 조절 및 코딩 서열을 포함하는 모든 유전자를 의미한다. 따라서, 키메라 유전자는 다른 공급원으로부터 유래된 조절 서열 및 코딩 서열, 또는 동일한 공급원으로부터 유래되었으나 자연에서 발견되는 것과는 다른 방식으로 배열된 조절 서열 및 코딩 서열을 포함할 수 있다. 유사하게, "외부" 유전자는 숙주 유기체에서 일반적으로 발견되지 않지만 유전자 전달에 의해 숙주 유기체 내로 도입되는 유전자를 지칭한다. 외래 유전자에는 비원래 유기체에 삽입된 고유 유전자 또는 키메라 유전자가 포함된다.
인-프레임(In-Frame): 본 출원에서 용어 "인프레임", 특히 "인프레임 융합 폴리뉴클레오타이드(in-frame fusion polynucleotide)"라는 문구는 업스트림 또는 5' 폴리뉴클레오타이드 또는 폴리뉴클레오타이드에 있는 코돈의 리딩 프레임과 동일한 리딩 프레임인 ORF 또는 상류 폴리뉴클레오타이드의 하류 또는 3'에 위치한 ORF 또는 업스트림 또는 5' 폴리뉴클레오타이드 또는 ORF와 융합된 ORF에 있는 코돈의 판독 프레임을 의미한다. 이러한 인프레임 융합 폴리뉴클레오타이드는 5' 폴리뉴클레오타이드와 3' 폴리뉴클레오타이드 모두에 의해 코딩되는 융합 단백질 또는 융합 펩타이드를 코딩한다.
시험관 내 전사 반응(In vitro transcription reaction): 본 명세서에 사용된 "시험관내 전사 반응"은 시험관내에서 DNA 주형을 전사함으로써 RNA를 생성하도록 고안된 반응이다. 시험관 내 전사 반응에는 전사될 RNA를 코딩하는 하나 이상의 DNA 주형 분자, 하나 이상의 완전히 또는 부분적으로 정제된 단일-소단위 RNA 중합효소, 단일-소단위 RNA 중합효소(들)에 대한 기질로서 최소 4개의 뉴클레오사이드 트리포스페이트, 반응에 필요한 완충액, 2가 양이온 및 염이 포함되어 있다.
반복/반복적(Iterate/Iterative): 본 출원에서 반복한다는 것은 재료나 샘플에 방법이나 절차를 반복적으로 적용하는 것을 의미한다. 일반적으로 각 처리, 변경 또는 수정 라운드에서 생성된 처리, 변경 또는 수정된 재료 또는 샘플은 다음 라운드의 처리, 변경 또는 수정을 위한 출발 물질로 사용된다. 반복 선택은 한 라운드 선택의 생존자를 다음 라운드의 시작 자료로 사용하여 선택을 두 번 이상 반복하거나 반복하는 선택 프로세스를 나타낸다.
라이브러리: 유전자 또는 폴리뉴클레오타이드 서열의 라이브러리는 서로 다르며 서열 전파를 위해 벡터에 클로닝된 서열의 모음이다. 다양한 라이브러리에서, 서열은 서열 내용, 기원, 근원 유기체, 길이, 구조, 다른 서열과의 연관 및/또는 폴리뉴클레오타이드 서열의 기타 특성에 따라 다르다. 예를 들어, 아미노산 반복 융합 유전자의 라이브러리는 E. coli 게놈에 의해 인코딩된 여러 개의 서로 다른 ORF를 포함하는 시작 ORF 컬렉션(starting ORF collection)을 박테리아 클로닝에 클로닝 및 프로모터, 이 서열이 ORF에 인프레임으로 직접 연결되는 방식으로 배향된 아미노산 반복을 코딩하는 서열, 터미네이터, 플라스미드 백본 및 항생제 내성 유전자를 포함하는 발현 벡터에 의하여 생성된다. 시작 ORF 컬렉선에는 5개 또는 그 이상, 예를 들어 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 5 0000, 60000, 70000, 80000, 90000, 100000 이상 또는 그 사이의 수의 ORF가 포함될 수 있다. 본 개시내용의 특정 측면에서, 라이브러리를 생성하는데 사용된 ORF 컬렉션에는 E. coli의 특정 바람직한 특성을 인코딩할 가능성이 높을 만큼 충분한 수의 ORF, 예를 들어 E. coli 게놈에 의해 암호화된 ORF의 50% 이상, 또는 2074 또는 총 4148개의 ORF를 나열하는 매디슨 위스콘신 대학에서 준비한 E. coli 균주 MG1655 게놈 주석의 주석을 사용할 때 더 많은 ORF가 포함되어 있다.
링커 순서: 이 문구는 융합 폴리뉴클레오타이드 또는 융합 폴리펩티드에서 2개의 폴리뉴클레오타이드 또는 폴리펩티드를 분리하는 폴리뉴클레오타이드 서열 또는 폴리펩티드 서열을 의미한다. 예를 들어, 융합 폴리뉴클레오타이드는 링커 서열에 의해 분리된 2개 이상의 ORF를 함유하며, 이는 융합 폴리뉴클레오타이드의 발현 및 번역의 결과인 폴리펩티드의 두 부분을 분리하는 펩티드를 코딩한다. 링커는 단백질이나 효소로부터 에피토프 태그를 분리할 수도 있다. 링커 서열은 다양한 길이 및/또는 서열 구성을 가질 수 있다.
비상동성(Non-homologous): 본 출원에서 용어 "비상동성"은 50% 미만의 뉴클레오타이드 수준에서 서열 동일성을 갖는 것으로 정의된다.
핵산: 핵산이라는 용어는 포스포디에스테르 결합, 포스포로티오에이트 결합 또는 기타 결합을 통해 서로 결합된 뉴클레오타이드로 구성된 생체고분자를 의미한다. "핵산" 또는 "핵산 분자"는 폴리뉴클레오타이드와 상호교환적으로 사용될 수 있다. 본 명세서에서 사용되는 용어 핵산은 단일 가닥의 핵산을 의미한다. 핵산은 디옥시리보뉴클레오타이드 잔기(이 경우 DNA) 또는 리보뉴클레오타이드 잔기(이 경우 RNA)로 구성될 수 있고, 또는 이는 데옥시리보뉴클레오타이드 잔기와 리보뉴클레오타이드 잔기를 모두 포함할 수 있으며, 이 경우 이는 키메라 핵산이다.
핵산 기질 또는 기질 핵산 분자: 이는 핵산 중합효소에 의해 촉매되고 뉴클레오사이드 트리포스페이트를 뉴클레오타이드 공급원으로 사용하는 반응 동안 뉴클레오타이드 수용체 역할을 하는 효소적 뉴클레오타이드 첨가 반응 또는 효소적 핵산 합성 반응에 존재하는 핵산 분자이다. 예를 들어, 효소와 하나 이상의 데옥시뉴클레오사이드 트리포스페이트의 존재 하에 반응된 단일 가닥 DNA 올리고뉴클레오타이드는 이 반응에서 기질 핵산 분자이다.
핵산 중합효소: 뉴클레오사이드 트리포스페이트와 비차단 핵산을 기질로 사용하여 핵산의 중합을 촉매하고, 비차단 핵산의 3' 말단에 단일 뉴클레오타이드를 순차적으로 첨가하는 효소이다. 과학 문헌에 설명된 핵산 중합효소는 일반적으로 DNA 중합효소와 RNA 중합효소의 부류에 속하며, DNA 중합효소는 DNA를 중합할 수 있고 RNA 중합효소는 RNA를 중합할 수 있다. 그러나 특정 효소는 DNA와 RNA의 합성을 모두 촉매하는 이중 능력을 가질 수 있다. 예를 들어, DNA 중합효소는 DNA 또는 RNA 분자의 3' 말단에 리보뉴클레오타이드를 추가하는 능력이 있을 수 있고, RNA 중합효소는 DNA 또는 RNA 분자의 3' 말단에 데옥시리보뉴클레오타이드를 추가하는 능력이 있을 수 있다.
핵산 합성: 이는 핵산 중합효소, 단량체 빌딩 블록인 하나 이상의 뉴클레오사이드 트리포스페이트 및 핵산 기질을 최소한으로 필요로 하는 자연에서 또는 인간에 의해 핵산이 생산되는 과정이다.
De novo 핵산 합성: 이는 핵산의 특정 서열과 구조를 생성하기 위해 핵산 기질에 특정 뉴클레오타이드를 조절하여 첨가하는 것을 포함하는 인공 DNA의 합성을 가리키는 데 사용된다.
뉴클레오타이드: 이는 5탄당, 인산기 및 질소 염기의 세 가지 구성 요소로 구성된 핵산의 단량체 구성 요소이다. 뉴클레오타이드의 두 가지 주요 클래스는 DNA의 구성 요소인 디옥시리보뉴클레오타이드와 RNA의 구성 요소인 리보뉴클레오타이드다. 당이 리보스라면 핵산은 RNA이고, 당이 리보스 유도체 디옥시리보스라면 핵산은 DNA이다. 본 명세서에 사용된 바와 같이, 데옥시리보뉴클레오타이드는 리보스 당의 2' 탄소로서 CH2 그룹을 갖는다. 2' 탄소의 다른 모든 구조는 리보뉴클레오타이드라는 용어로 분류된다. 본 명세서에 사용된 바와 같이, 뉴클레오타이드는 핵산, 뉴클레오사이드 모노포스페이트, 뉴클레오사이드 디포스페이트, 뉴클레오사이드 트리포스페이트 또는 이들의 임의의 유도체 또는 변형 내에 존재하는 뉴클레오타이드 잔기를 의미할 수 있다.
뉴클레오사이드 트리포스페이트: 본 출원에서 "뉴클레오사이드 트리포스페이트"는 RNA 합성에 사용되는 리보뉴클레오사이드 트리포스페이트 ATP, CTP, GTP, ITP, UTP 및 XTP 등 중 하나 또는 데옥시리보뉴클레오사이드 트리포스페이트 dATP, dCTP, dGTP, dITP, dTTP 및 DNA 합성에 사용되는 dXTP 등 중 하나, 또는 포스포로티오에이트 결합을 함유하는 유도체를 포함하는 이의 변형된 유사체, 유도체 또는 변이체로 정의된다. DNA 합성에 사용되는 4가지 표준 뉴클레오사이드 트리포스페이트(dATP, dCTP, dGTP 및 dTTP)의 혼합물은 약어로 "dNTP"로 표시되며, RNA 합성에 사용되는 4가지 표준 뉴클레오사이드 트리포스페이트(ATP, CTP, GTP 및 UTP)의 혼합물은 약어로 "NTP"로 표시된다.
올리고뉴클레오타이드: 올리고뉴클레오타이드라는 용어는 2개 이상의 뉴클레오타이드로 구성된 단일 가닥 핵산을 의미한다.
오픈 리딩 프레임(ORF): ORF는 특정 리딩 프레임의 코돈 문자열로 단백질이나 펩타이드를 암호화하는 핵산의 뉴클레오타이드 서열로 정의된다. 이 특정 리당 프레임 내에서 ORF는 아미노산을 지정하는 모든 코돈을 포함할 수 있지만 정지 코돈은 포함하지 않는다. 시작 컬렉션(starting collection)의 ORF는 특정 아미노산으로 시작하거나 끝날 필요가 없다. ORF는 연속적이거나 하나 이상의 인트론에 의해 중단된다.
작동 가능하게 연결됨(Operably linked): "작동 가능하게 연결됨"이라는 용어는 하나의 기능이 다른 하나의 기능에 영향을 받도록 단일 핵산 단편에 대한 핵산 서열의 결합을 의미한다. 예를 들어, 프로모터는 코딩 서열의 발현에 영향을 미칠 수 있는 경우(즉, 코딩 서열이 프로모터의 전사 제어 하에 있음) 코딩 서열과 작동가능하게 연결된다. 코딩 서열은 센스 또는 안티센스 방향으로 조절 서열에 작동가능하게 연결될 수 있다.
펩티드 결합: "펩티드 결합"은 첫 번째 아미노산의 알파-아미노기가 두 번째 아미노산의 알파-카르복실기에 결합되어 있는 첫 번째 아미노산과 두 번째 아미노산 사이의 공유 결합이다.
서열 동일성 백분율(Percentage of sequence identity): "백분율 서열 동일성"이라는 용어는 임의의 주어진 쿼리 서열, 예를 들어 서열 번호 10과 대상 서열 사이(subject sequence)의 동일성 정도를 지칭한다. 대상 서열은 일반적으로 쿼리 시퀀스 길이의 약 80% 내지 200%인 길이(예를 들어, 쿼리 시퀀스 길이의 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190 또는 200%)이다. 쿼리 핵산 또는 폴리펩티드에 대한 임의의 대상 핵산 또는 폴리펩티드의 동일성 백분율은 다음과 같이 결정된다. 쿼리 서열(예를 들어, 핵산 또는 아미노산 서열)은 컴퓨터 프로그램 ClustalW (version 1.83, default parameters)를 사용하여 하나 이상의 대상 핵산 또는 아미노산 서열에 정렬되며, 이는 핵산 또는 단백질 서열의 정렬이 전체 길이에 걸쳐 수행될 수 있게 한다 (global alignment, Chenna 2003).
쿼리 서열에 대한 대상체 또는 핵산 또는 아미노산 서열의 동일성 백분율을 결정하기 위해, Clustal W를 사용하여 시퀀스를 정렬하고, 정렬에서 동일한 일치 항목 수를 쿼리 길이로 나누고 결과에 100을 곱한다. 백분율 동일성 값은 가장 가까운 10분의 1로 반올림될 수 있다는 점에 유의한다. 예를 들어, 78.11, 78.12, 78.13, 78.14는 78.1로 반올림되고, 78.15, 78.16, 78.17, 78.18, 78.19는 78.2로 반올림된다.
ClustalW는 쿼리와 하나 이상의 대상 서열 간에 최상의 매치를 계산하고, 이들을 정렬하여 상동성, 유사성 및 차이점들을 확인할 수 있도록 해준다. 서열 정렬을 극대화하기 위해, 일 이상의 잔기의 간극(gaps)이 쿼리 서열, 대상 서열, 또는 둘 모두에 삽입될 수 있다. 핵산 서열을 빠르게 쌍으로 정렬하기 위해 기본 매개 변수가 사용될 수 있다(즉, 단어 크기(word size): 2; 창크기(window size): 4; 스코어링 방법(scoring method): 퍼센트; 꼭대기 사선의 수(number of top diagonals): 4; 및 간극 페널티(gap penalty): 5. 핵산 서열의 다중 정렬을 위해 다음의 매개 변수가 사용될 수 있다: 간극 오프닝 페널티(gap opening penalty): 10.0; 간극 확장 페널티(gap extension penalty): 5.0; 및 웨이트 트랜지션(weight transitions): 있음(yes). 폴리펩티드 서열을 빠르게 쌍으로 정렬하기 위해, 다음의 매개 변수가 사용될 수 있다: 단어 크기(word size): 1; 창크기(window size): 5; 스코어링 방법(scoring method): 퍼센트; 꼭대기 사선의 수(number of top diagonals): 5; 및 간극 페널티(gap penalty): 3. 폴리펩티드 서열의 다중 정렬을 위해 다음의 매개 변수가 사용될 수 있다: 웨이트 매트릭스(weight matrix): blosum; 간극 오프닝 페널티(gap opening penalty): 10.0; 간극 확장 페널티(gap extension penalty): 0.05; 친수성 간극(hydrophilic gaps): on; 친수성 잔기(hydrophilic residues): Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, 및 Lys; 및 잔기-특이적 간극 페널티(residue-specific gap penalties): on. ClustalW 출력은 시퀀스 간의 관계를 반영하는 시퀀스 정렬이다. 예를 들어, ClustalW는 월드와이드웹(World Wide Web)의 베일러 대학 의학 연구 런처(the Baylor College of Medicine Search Launcher) 웹사이트 또는 유럽 바이오인포매틱스 연구원(the European Bioinformatics Institute) 웹사이트에서 가동할 수 있다.
플라스미드 및 벡터: "플라스미드" 및 "벡터"라는 용어는 세포나 유기체의 자연적인 부분이 아닌 유전자를 전달하는데 사용되는 유전 요소를 의미한다. 플라스미드는 일반적으로 자율 에피솜 유전 요소(autonomous episomal genetic element)로서 염색체 외적으로 복제되는 반면, 벡터는 게놈에 통합되거나 선형 또는 원형 DNA 단편으로 염색체 외에서 유지될 수 있다. 플라스미드와 벡터는 선형 또는 원형일 수 있으며 모든 소스에서 파생된 단일 및/또는 이중 가닥 DNA 또는 RNA로 구성될 수 있다. 플라스미드 및 벡터는 종종 폴리뉴클레오타이드 서열을 세포 또는 유기체에 도입하고 유기체 내에서 유전자를 발현하는데 유용한 독특한 구성으로 결합되거나 재조합된 다양한 소스로부터의 다수의 뉴클레오타이드 서열을 포함한다. 플라스미드 또는 벡터에 존재하는 서열에는 다음이 포함되지만 이에 국한되지는 않는다: 자율적 복제 서열(autonomously replicating sequences); 동원체 서열(centromere sequences); 게놈 통합 서열(genome integrating sequences); 복제 기원(origins of replication); 프로모터 및/또는 터미네이터와 같은 제어 서열(control sequence); 오픈 리딩 프레임(open reading frame); 항생제 내성 유전자와 같은 선택 가능한 마커 유전자; 형광 단백질을 코딩하는 유전자와 같은 가시적 마커 유전자; 제한 엔도뉴클레아제 인식 부위(endonuclease recognition site); 재조합 사이트; 및/또는 명백하거나 알려진 기능이 없는 서열.
폴리펩티드 또는 단백질: "폴리펩티드" 또는 "단백질"이라는 용어는 펩티드 결합으로 연결된 복수의 아미노산 단량체로 구성된 중합체를 의미한다. 폴리머는 10개 또는 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 또는 그 사이의 숫자를 포함하는 이상의 모노머를 포함한다.
프로모터: "프로모터"라는 용어는 코딩 서열 또는 기능적 RNA의 발현을 제어할 수 있는 DNA 서열을 의미한다. 일반적으로 코딩 서열은 프로모터 서열의 3'에 위치한다. 프로모터는 그 전체가 천연 유전자로부터 유래될 수 있고/있거나 자연에서 발견되는 다양한 프로모터로부터 유래된 다양한 요소로 구성될 수 있거나 심지어 합성 DNA 세그먼트를 포함할 수도 있다. 당업자는 다양한 프로모터가 다양한 조직 또는 세포 유형에서, 또는 다양한 발생 단계에서, 또는 다양한 환경 또는 생리학적 조건에 반응하여 유전자의 발현을 지시한다는 것을 이해한다. 대부분의 세포 유형에서 대부분의 경우 유전자가 발현되도록 하는 프로모터를 일반적으로 "구성적 프로모터"라고 한다. 대부분의 경우 조절 서열의 정확한 경계가 완전히 정의되지 않았기 때문에 길이가 다른 DNA 단편이 동일한 프로모터 활성을 가질 수 있다는 것이 추가로 인식된다.
무작위/무작위화(Random/Randomized): 본 명세서에 사용된 바와 같이, 방법이나 의식적인 결정 없이 만들어지거나 선택되는 것을 의미한다.
RNA: "RNA"는 리보뉴클레오타이드의 중합체인 핵산이다. RNA는 단일 가닥 또는 이중 가닥 형태로 발생한다. 본 명세서에 사용된 바와 같이, RNA는 각각 CH2가 아닌 형태의 2' 탄소를 갖는 뉴클레오타이드 잔기를 포함한다.
서열: 당업자에게 알려진 바와 같이, 생물학적 맥락에서 사용될 때 "서열"은 핵산의 뉴클레오타이드 서열 또는 단백질의 아미노산 서열을 의미할 수 있다. 본 명세서에 사용된 용어 "서열"은 해당 용어가 사용되는 문맥에 따른 의미를 갖는다. 예를 들어, 게놈 서열, 유전자 서열 또는 ORF와 같은 핵산을 암시하는 맥락에서 사용되는 경우, 서열은 뉴클레오타이드 서열을 의미한다. 프로테옴, 단백질 또는 효소와 같은 단백질 또는 폴리펩티드를 암시하는 문맥에서, 서열은 아미노산 서열을 의미한다.
서열 특이적 뉴클레오타이드 첨가: 본 명세서에 사용된 바와 같이, 이는 활성에서 서열 특이성을 나타내는 핵산 중합효소의 특징이다. 예를 들어, 주형 독립적 DNA 중합효소는 dT 잔기로 끝나는 핵산의 3' 말단에만 뉴클레오타이드를 추가할 수 있고 다른 뉴클레오타이드로 끝나는 3' 말단에는 추가할 수 없는 서열 특이성을 가질 수 있다. 핵산 중합효소의 이러한 서열 특이성은 부분적이거나 완전할 수 있다. 부분적이라면, 위 예의 DNA 중합효소는 3' dT 잔기로 끝나는 핵산에 뉴클레오타이드를 더 효율적으로 추가할 것이지만, 비록 덜 효율적이긴 하지만 3' dA, dC 또는 dG 잔기로 끝나는 핵산도 변형할 것이다. 완료되면 위 예의 DNA 중합효소는 3' dT 잔기로 끝나는 핵산에만 뉴클레오타이드를 추가하고 3' dA, dC 또는 dG 잔기로 끝나는 핵산은 변형하지 못한다.
주형 독립적 핵산 중합효소(Template-independent nucleic acid polymerase): "주형 독립적 핵산 중합효소"는 무기 인산염의 방출을 동반하며, 합성되는 가닥에 염기쌍을 이루고 합성되는 가닥에 대한 주형 역할을 하는 또 다른 핵산 가닥이 없는 경우, 핵산의 3'-히드록실 말단에 뉴클레오타이드의 통합을 촉매하는 효소이다. 구체적으로, 주형 독립적 DNA 중합효소는 주형을 사용하지 않고 DNA 가닥의 중합을 촉매하는 반면, 주형 독립적 RNA 중합효소는 주형을 사용하지 않고 RNA 가닥의 중합을 촉매한다.
주형 독립적 핵산 합성(Template-independent Nucleic Acid Synthesis): 이는 합성되는 핵산과 염기쌍을 이루고 합성되는 가닥의 주형 역할을 하는 주형 가닥을 사용하지 않고 핵산 중합효소가 핵산의 중합을 촉매하는 과정이다.
형질전환된(Transformed): 용어 "형질전환된"은 폴리뉴클레오타이드 서열의 도입에 의한 유전적 변형을 의미한다.
형질전환: 본원에 사용된 용어 "형질전환"은 핵산 단편이 숙주 유기체로 전달되어 유전적으로 안정한 유전을 초래하는 것을 의미한다. 형질전환된 핵산 단편을 함유하는 숙주 유기체는 "형질전환(transgenic)" 또는 "재조합" 또는 "형질전환" 유기체로 지칭된다.
형질전환된 유기체(Transformed Organism): 형질전환된 유기체는 폴리뉴클레오타이드 서열을 유기체의 게놈에 도입함으로써 유전적으로 변경된 유기체이다.
전위(Translocation): 핵산 중합효소의 "전위"는 핵산 기질에 뉴클레오타이드를 첨가한 후 핵산 중합 방향(5'에서 3')으로 핵산 주형을 따라 효소가 이동하는 것을 의미한다. 핵산 중합효소는 기질에 뉴클레오타이드를 첨가한 후 주형이나 핵산 기질을 따라 이동한다.
불리한 조건(Unfavorable Condition): 본 명세서에 사용된 바와 같이, 이 문구는 정상적인 성장 조건에서보다 느린 성장을 초래하거나 정상적인 성장 조건에 비해 세포의 생존력을 감소시키는 물리적 또는 화학적 성장 조건의 모든 부분을 의미한다.
차단되지 않은 핵산(Unblocked Nucleic Acid): 이 문구는 유리 3' 수산기를 갖는 핵산을 의미한다.
차단되지 않은 뉴클레오타이드 또는 차단되지 않은 뉴클레오사이드 트리포스페이트 또는 차단되지 않은 dNTP 또는 차단되지 않은 NTP: 이 문구는 상호 교환적으로 사용되며 유리 3' 하이드록실 그룹이 있는 뉴클레오타이드 또는 뉴클레오사이드 트리포스페이트를 나타낸다.
본 개시내용에서 용어 "인프레임", 특히 "인프레임 융합 폴리뉴클레오타이드(in-frame fusion polynucleotide)"라는 문구는 업스트림 또는 5' 폴리뉴클레오타이드, 폴리뉴클레오타이드의 코돈 리딩 프레임과 동일한 유전자 또는 ORF, 상류 폴리뉴클레오타이드의 하류 또는 3'에 위치하는 유전자 또는 ORF, 상류 또는 5' 폴리뉴클레오타이드와 융합되는 유전자 또는 ORF, 유전자 또는 ORF에 있는 코돈의 판독 프레임을 의미한다. 이러한 인프레임 융합 폴리뉴클레오타이드의 집합은 서로에 대해 인프레임인 업스트림 및 다운스트림 폴리뉴클레오타이드를 함유하는 융합 폴리뉴클레오타이드의 백분율이 다양할 수 있다. 전체 컬렉션의 비율은 최소 10%이며 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% 또는 그 사이의 숫자가 가능하다.
XTP 또는 dXTP: 용어 "XTP" 또는 "dXTP"는 RNA 합성에 사용되는 임의의 리보뉴클레오사이드 트리포스페이트 또는 자연 발생 리보뉴클레오사이드 트리포스페이트의 임의의 변형된 형태 또는 RNA의 변형된 형태, 또는 DNA 합성에 사용되는 임의의 데옥시리보뉴클레오사이드 트리포스페이트 또는 자연 발생 데옥시리보뉴클레오사이드 트리포스페이트의 임의의 변형된 형태 또는 DNA의 변형된 형태를 의미한다.
본 개시내용은 주형-독립적인 방식(template-independent manner)으로 핵산을 합성하기 위한 조성물 및 방법을 제공한다. 특정 핵산 중합효소는 추가할 뉴클레오타이드 유형이나 추가를 안내하는 주형 없이 핵산의 자유 3' 말단에 뉴클레오타이드를 추가하는 능력이 있다. 본 개시내용에서 이러한 중합효소는 주형 독립적 핵산 중합효소(template-independent nucleic acid polymerase, TINAP) 활성을 갖는 것으로 지칭된다.
TINAP 활성을 갖는 중합효소는 시험관 내에서 인공 핵산을 생성하는데 유용하다. 예를 들어,TINAP 활성을 갖는 핵산 중합효소는 핵산 합성을 허용하는 실험 조건 하(예를 들어, 생리학적 pH, 완충제 및 2가 양이온 보조인자의 존재 하, 핵산 중합을 허용하는 온도에서 배양)에서 하나 이상의 뉴클레오사이드 트리포스페이트(nucleoside triphosphate) 및 유리 3' 수산기(free 3' hydroxyl group)를 포함하는 하나 이상의 기질 핵산(substrate nucleic acid)과 결합될 수 있다. 상기 중합효소는 단일 추가 주기(single addition cycle)에서 기질 핵산의 3' 말단이 단일 뉴클레오타이드에 의해 연장되는 방식으로 3' 말단에 대한 뉴클레오타이드 추가를 촉매한다. 그런 다음 핵산 분자는 효소 및/또는 뉴클레오사이드 트리포스페이트로부터 분리되고 주기가 반복된다. 이러한 방식으로, 임의의 특정 핵산 서열은 한 번에 하나의 뉴클레오타이드씩 순환(one nucleotide at a time) 방식으로 합성될 수 있다.
위에 설명된 전략에서 특정 핵산 서열을 합성하는 능력은 첨가 주기(addition cycle)당 단일 뉴클레오타이드만큼 기질 핵산을 확장하는 TINAP 활성을 갖는 핵산 중합효소의 능력에 따라 달라진다. 핵산 중합효소의 작은 하위 집합(subset)에는 이러한 능력이 있다.
현재까지, 한 번에 하나의 뉴클레오타이드를 합성할 수 있는 EOS 전략을 개발하려는 다른 노력에서는 핵산에 추가되는 뉴클레오타이드의 3' 하이드록실에 공유 결합된 화학 그룹을 포함하는 3' 차단된 뉴클레오타이드를 사용해야 했다. 3' 하이드록실을 변형하는 화학적 차단 그룹은 기질 핵산 분자의 유리 3' 하이드록실 그룹에 여러 뉴클레오타이드가 추가되는 것을 방지한다. 한 차례의 첨가 후, 상기 핵산 기질 분자는 효소와 뉴클레오사이드 트리포스페이트로부터 분리되고 화학적 차단 그룹은 기질 핵산 분자의 나머지 부분을 변경하지 않고 그대로 두는 처리를 통해 제거된다. 3' 히드록실은 이 차단 해제 단계 동안 노출되어 또 다른 추가 주기를 위해 기질 핵산 분자를 준비한다. 이 전략은 그림 1A에 나와 있다.
본 개시내용에 기술된 EOS 전략은 차단되지 않거나 유리된 3' 하이드록실을 갖는 천연 뉴클레오타이드를 사용하는 것으로 3'-차단된 뉴클레오타이드를 사용하여 위에 기술된 것과 상이하다. 본 개시내용에서 추가 주기당 단일 뉴클레오타이드의 첨가는 추가 주기당 단일 뉴클레오타이드로 기질 핵산 분자를 확장할 수 있게 하는 TINAP 활성을 갖는 핵산 중합효소의 특정 품질에 따라 달라진다. 본 개시내용에 설명된 EOS 전략은 도 1C에 예시되어 있다.
본 개시내용에 기술된 전략에 기초한 핵산 합성 공정은 중합효소 활성에 적합한 반응 혼합물 (생리학적 pH 또는 그에 가까운 완충제 및 2가 양이온을 최소한으로 포함)에서 기질 핵산 분자, 핵산 중합효소(TINAP) 및 하나 이상의 뉴클레오사이드 트리포스페이트를 혼합(combining)하는 단계, 반응이 완료될 때까지 충분한 시간 동안 반응이 진행되도록 반응을 허용(allowing)하는 단계; 그런 다음 단일 뉴클레오타이드의 첨가에 의해 변형된 기질 핵산 분자를 핵산 중합효소 및 통합되지 않은(unincorporated) 뉴클레오사이드 트리포스페이트로부터 분리(separating)하는 단계, 및 상기 주기를 반복(repeating)하는 단계를 최소한으로 포함한다.
본 개시내용은 핵산 합성을 위한 임의의 차단되지 않은 뉴클레오사이드 트리포스페이트의 사용을 포함한다. 뉴클레오사이드 트리포스페이트는 RNA 또는 RNA의 변형된 형태를 합성하는데 사용되는 ATP, CTP, GTP, ITP, UTP 또는 XTP와 같은 리보뉴클레오사이드 트리포스페이트 또는 이들의 임의의 변형된 형태일 수 있다. 뉴클레오사이드 트리포스페이트는 DNA 또는 DNA의 변형된 형태를 합성하는데 사용되는 dATP, dCTP, dGTP, dITP, dUTP 또는 dXTP와 같은 데옥시리보뉴클레오사이드 트리포스페이트 또는 그의 임의의 변형된 형태일 수 있다.
뉴클레오타이드의 변형된 형태는 메틸기, O-메틸기, 히드록실기, 아미노기, 인산염, 염소 또는 불소 원자, 단당류, 이당류 또는 다당류, 염료, 형광기(fluorescent group), 포스포로티오에이트 기(phosphorothioate group)(포스포디에스테르 결합의 산소 원자를 황 원자로 치환), 결합기(예를 들어 비오틴 또는 디옥시게닌), 아지드, 알데히드, 케톤, 티올, 이황화물 또는 아민과 같은 반응성 기, 또는 상기 중 하나 이상을 포함하는 분자를 포함하지만, 이에 제한되지 않는다. 변형기(Modifying group)는 뉴클레오타이드의 질소 염기 또는 리보스 당의 2' 또는 5' 탄소(예를 들어 2'-플루오로 또는 2'-O-메틸 치환)에 추가될 수 있지만 3'-하이드록실 그룹을 제외하고 뉴클레오타이드에서 발견되는 모든 탄소, 질소 또는 산소 원자를 변형할 수 있다. 단일 뉴클레오타이드 분자에 여러 변형기를 추가할 수 있다. 상기 뉴클레오타이드에 추가된 변형기의 목적은 변형된 뉴클레오타이드가 공유적으로 추가된 분자의 특정 검출, 정제, 표적화(유기체의 조직 또는 세포 유형에 대한) 또는 이들의 조합을 허용하는 것이다.
본 개시내용은 임의의 서열의 임의의 핵산 분자를 합성하는데 사용될 수 있다. 합성된 핵산 분자는 DNA 또는 RNA 또는 이의 변형된 형태, 또는 리보뉴클레오타이드와 데옥시리보뉴클레오타이드 또는 이의 변형된 형태를 모두 포함하는 키메라 핵산일 수 있다. 합성된 서열은 2'-플루오로 또는 2'-O-메틸 치환을 포함하지만 이에 국한되지 않는 리보스 당에 대한 다양한 변형과 함께 표준 리보스 또는 데옥시리보스 백본 또는 이의 변형된 형태를 포함할 수 있다. 합성된 서열은 DNA 및 RNA에서 발견되는 표준 염기(아데닌, 시티딘, 구아닌, 티민, 우라실) 또는 흔하지 않은 염기(예를 들어 하이포잔틴, 크산틴) 또는 이러한 염기의 변형된 형태, 또는 천연 또는 변형된 염기의 임의의 혼합물을 포함할 수 있다. 질소성 염기(nitrogenous base)의 변형된 형태에는 메틸기, O-메틸기, 히드록실기, 아미노기, 인산염, 염소 또는 불소 원자, 단당류, 이당류 또는 다당류, 염료, 형광기(fluorescent group), 포스포로티오에이트 기(phosphorothioate group)(포스포디에스테르 결합의 산소 원자를 황 원자로 치환), 결합기(binding group, 예를 들어 비오틴 또는 디옥시게닌), 아지드, 알데히드, 케톤, 티올, 이황화물 또는 아민과 같은 반응성 기, 또는 상기 중 하나 이상을 포함하는 분자를 포함하지만, 이에 제한되지 않는다.
효소적 핵산 합성 반응에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 임의의 길이나 서열을 가질 수 있다. 예를 들어, 기질 핵산 분자는 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 또는100000개 이상의 뉴클레오타이드 또는 그 사이의 임의 길이일 수 있다.
효소적 핵산 합성 반응에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 용액 상태로 존재할 수도 있고, 아가로스 비드, 폴리스티렌 비드 또는 자기 비드와 같은 고체 지지체에 고정될 수도 있다. 기질 핵산 분자의 고정화는 고체 지지체에 대한 공유 결합을 통해 또는 고체 지지체와의 비공유 결합을 통해 발생할 수 있다.
효소적 핵산 합성 반응(enzymatic nucleic acid synthesis)에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 단일 가닥이거나 부분적으로 단일 가닥일 수 있다. 뉴클레오타이드 수용체 역할을 하는 기질 핵산 분자의 3' 말단은 단일 가닥으로, 즉, 이는 상동성 뉴클레오타이드와 염기쌍을 이루지 않지만, 3' 말단의 5'에 있는 기질 핵산 분자의 모든 뉴클레오타이드는 단일 가닥 또는 이중 가닥일 수 있다.
효소적 핵산 합성 반응에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 또는 100000 개 이상의 뉴클레오타이드 또는 그 사이의 임의 길이를 포함하는 임의의 길이일 수 있다.
효소적 핵산 합성 반응에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 데옥시리보뉴클레오타이드 잔기 또는 리보뉴클레오타이드 잔기, 또는 데옥시리보뉴클레오타이드와 리보뉴클레오타이드 잔기 둘 다의 혼합물을 함유할 수 있다. 기질 핵산 분자의 뉴클레오타이드 잔기는 리보스 당에 대한 변형, 염기에 대한 변형, 또는 백본에 대한 변형을 포함하여 임의의 변형을 함유할 수 있다.
효소적 핵산 합성 반응에서 뉴클레오타이드 수용체로 사용되는 기질 핵산 분자는 특정 서열 및 구조의 순수한 분자일 수 있거나, 다양한 서열 또는 구조의 혼합 집단일 수 있다.
본 개시내용에 기술된 조성물 및 방법을 사용하여 합성된 핵산 서열은 합성된 유형의 핵산(즉, DNA의 경우 A, C, G 및 T)에서 일반적으로 발견되는 모든 염기 또는 이러한 염기의 하위 집합을 포함할 수 있다. 상기 합성된 서열은 복잡하거나 비반복적일 수 있거나, 하나 이상의 특정 서열이 반복되는 반복적일 수 있다. 상기 합성된 서열은 동종중합체(homopolymeric)(단일 뉴클레오타이드만 함유)일 수 있거나, 반복 길이당 2개 이상의 뉴클레오타이드로 구성된 단순 반복, 또는 길이가 5개 이상의 뉴클레오타이드로 구성된 복합 반복을 포함할 수 있다.
본 개시내용에 기술된 조성물 및 방법을 사용하여 합성된 핵산 분자는 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 또는 100000 뉴클레오타이드 이상 또는 그 사이의 모든 길이를 포함하여 2 이상의 뉴클레오타이드 길이를 포함할 수 있다.
본 개시내용에 기술된 조성물 및 방법을 사용하여 핵산을 합성할 때 뉴클레오타이드 첨가 효율은 1% 내지 100% 범위일 수 있다. 이는 단일 추가 주기 동안 핵산 기질 분자의 하위 집합만이 핵산 중합효소에 의한 추가 뉴클레오타이드에 의해 연장될 수 있음을 의미한다. 예를 들어, 임의의 특정 핵산 기질 분자에 대한 임의의 특정 뉴클레오타이드의 첨가 효율은 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 115, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 또는 100% 또는 그 사이의 임의의 백분율이다.
핵산 중합효소에 의한 뉴클레오타이드 첨가 효율은 첨가 반응에 존재하는 각각의 뉴클레오사이드 트리포스페이트의 농도, 효소 농도 및 효소 활성에 영향을 미치는 반응 조건을 포함하되 이에 국한되지 않는 반응의 여러 요인 또는 변수에 의해 영향을 받을 수 있다. 예를 들어, 특정 뉴클레오사이드 트리포스페이트의 농도를 높이면 해당 뉴클레오사이드 트리포스페이트의 통합 효율이 증가할 수 있다. 유사하게, 특정 뉴클레오사이드 트리포스페이트의 통합을 촉매하는 효소의 농도를 증가시키는 것은 뉴클레오사이드 트리포스페이트의 통합 빈도를 증가시킬 수 있다. 반응 혼합물과 반응 조건을 변경함으로써, 예를 들어 완충제(예를 들어 트리스, 인산나트륨 또는 칼륨, 아세트산나트륨 또는 칼륨, 카코딜산나트륨 또는 칼륨), 염, 2가 양이온(divalent cations) 및 반응 첨가제 또는 폴리에틸렌 글리콜, 폴리비닐피롤리돈, 글리세롤, 폴리아민, 디터전트(detergent), 계면활성제, 소 혈청 알부민, DNA 결합 단백질, 포름아미드를 포함하되 이에 국한되지 않는 안정화제 또는 펩타이드 또는 소분자와 같은 핵산 중합효소 활성에 영향을 미치거나 변형시키는 분자의 존재를 변화시킴으로써; 또는 완충제, 염, 2가 양이온, 뉴클레오사이드 트리포스페이트 및 폴리에틸렌 글리콜, 폴리비닐피롤리돈, 글리세롤, 폴리아민, 디터전트(detergent), 계면활성제, 소 혈청 알부민, DNA 결합 단백질, 포름아미드를 포함하되 이에 국한되지 않는 기타 반응 성분 또는 펩타이드 또는 소분자와 같은 핵산 중합효소 활성에 영향을 미치거나 변형시키는 분자의 농도를 변화시킴으로써 동일한 결과를 얻을 수 있다.
핵산 합성 공정의 반응 pH는 여러 pH 단위(예: pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 또는 10.0 또는 그 사이의 pH)만큼 생리학적 pH 주변에서 달라질 수 있다.
핵산 중합효소에 의한 뉴클레오타이드 첨가의 알려진 메커니즘을 바탕으로, TINAP가 다중 뉴클레오타이드의 프로세스적 추가(processive addition)를 거치지 않고 차단되지 않은 핵산(unblocked nucleic acid)의 3' 말단에 단일 뉴클레오타이드의 추가를 촉매할 수 있는 다양한 가능한 메커니즘이 있다. 여기에는 다음이 포함되지만 이에 제한되지는 않는다. 1) 핵산 중합효소는, 핵산 기질의 말단 염기(terminal base)를 포함하는, 특정 핵산 서열에 특이적일 수 있으며, 이 특정 서열을 포함하는 기질 분자(substrate molecule)에만 뉴클레오타이드를 추가할 수 있다. 뉴클레오타이드가 추가되면 최종 서열(end sequence)이 달라지며 상기 중합효소가 기질에 다른 뉴클레오타이드를 추가하지 못할 수도 있다. 2) 핵산 중합효소는 뉴클레오타이드 추가 메커니즘의 전위 단계(translocation step)에 결함이 있을 수 있으며, 이는 뉴클레오타이드 추가 및 피로인산염(pyrophosphate) 방출의 촉매 단계 후에 효소를 정지(stall)시켜 중합효소가 단일 뉴클레오타이드만 추가하도록 허용한다. 3) 핵산 중합효소는 핵산 분자의 말단과 공유 또는 비공유 방식으로 긴밀하게 결합되어 뉴클레오타이드 첨가 후 중합효소의 해리(dissociation)를 방지하고 중합효소의 다른 분자가 핵산의 3' 말단에 접근하는 것을 방지한다. 4) 핵산 중합효소는 단일 뉴클레오타이드를 추가한 후 촉매 활성을 잃어 추가 뉴클레오타이드를 추가할 수 없게 될 수 있다. 이러한 메커니즘과 효소 특성은 특정 핵산 중합효소에 개별적으로 또는 조합되어 나타날 수 있다.
핵산의 3' 말단에 뉴클레오타이드를 추가할 때(위에 나열된 단일 뉴클레오타이드 추가의 첫 번째 메커니즘) 서열 특이성을 나타내는 핵산 중합효소는 핵산의 서로 다른 부분에 위치한 서로 다른 수의 뉴클레오타이드를 인식하고 이에 대해 특이성을 가질 수 있다. 예를 들어, 핵산 중합효소는 핵산의 3' 말단에 존재하는 서열에 특이적일 수도 있고, 3' 말단에 존재하는 뉴클레오타이드를 포함하지 않는 내부 서열에 특이적일 수도 있다. 중합효소는 핵산의 3' 말단에 또는 내부적으로 존재하는1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 또는 그 이상의 뉴클레오타이드에 특이적일 수 있다. 핵산 내부의 특정 서열을 인식할 때, 핵산의 3' 말단으로부터의 거리는 길이가 다를 수 있으며, 예를 들어 핵산의 3' 말단으로부터 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50개 또는 그 이상의 뉴클레오타이드가 있을 수 있다. 핵산 중합효소의 서열 특이성을 지배하는 인식 서열은 또한 핵산 내의 하나 이상의 비연속 서열에 존재할 수도 있다.
핵산의 3' 말단에 단일 뉴클레오타이드를 첨가한 후 촉매 활성을 잃는 핵산 중합효소는 가역적 또는 비가역적 방식으로 이를 수행할 수 있다. 가역적이라면 pH 변화; 염, 2가 양이온, 피로포스페이트, 뉴클레오사이드 모노포스페이트, 뉴클레오사이드 디포스페이트, 뉴클레오사이드 트리포스페이트, 환원제, 또는 전술한 것의 조합의 농도 변화; 중합효소 농도의 변화; 구아니딘, 요소 또는 알코올과 같은 카오트로픽제(chaotropic agent)를 사용한 처리; 완전히 펼쳐진 후 다시 부분적 또는 완전한 재접힘 또는 중합효소의 활성을 회복시키는 당업자에게 공지된 임의의 다른 처리가 있다. 활성 손실이 되돌릴 수 없는 경우 이러한 치료는 중합효소 활성을 회복하지 못한다.
산업용 핵산 합성 공정에 사용되는 핵산 중합효소는 한 번 사용한 후 폐기하거나 계속 사용하기 위해 뉴클레오타이드 추가 주기 사이에 재활용할 수 있다. 핵산 중합효소는 임의 개수의 뉴클레오타이드 추가 주기, 예를 들어 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100주기 또는 그 사이 의 임의 횟수에 사용될 수 있다. 주기 사이에, 다음 뉴클레오타이드 추가 주기를 위해 준비하기 위하여, 핵산 중합효소는 친화성 크로마토그래피, 음이온 교환 크로마토그래피, 양이온 교환 크로마토그래피, 겔 여과 크로마토그래피, 역상 크로마토그래피 또는 한외여과를 포함하지만 이에 제한되지 않는 다양한 단백질 정제 방법을 통해 탈염, 농축 또는 다른 반응 성분으로부터 분리될 수 있다.
뉴클레오타이드 추가 주기 사이에, 다음 뉴클레오타이드 추가 주기를 준비하기 위하여 산업용 핵산 합성 공정에 사용되는 핵산 중합효소는 부분적으로 또는 완전히 펼쳐지거나 변성(단백질을 특징적인 3차원 구조에서 무작위 코일로 부분적으로 또는 완전히 전환하는 것을 의미함)될 수 있으며 원래의 3차원 구조로 다시 접힐 수 있다.
단일 뉴클레오타이드 추가 반응은 기질과 효소의 서로 다른 화학량론을 사용할 수 있으며, 이는 세 가지 범주로 분류된다: 1) 몰 과량(Molar excess)의 효소; 2) 등몰량(Equimolar amount)의 효소 및 기질 말단 및 3) 몰 과량의 핵산 기질 3' 말단. 효소가 몰 과량인 경우, 상기 효소는 핵산 기질 3' 말단의 농도와 비교하여 배수 초과를 나타내는 농도, 예를 들어 1.01x, 1.01x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x 또는 그 사이의 임의의 수/배 초과로 존재할 수 있다. 핵산 기질 또는 기질의 3' 말단(예를 들어 공유 고정된 기질의 경우)은 효소의 농도에 비해 배수 초과를 나타내는 농도, 예를 들어 1.01x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x 또는 그 사이의 임의의 수/배 초과로 존재할 수 있다.
단일 뉴클레오타이드의 첨가를 조절하여 핵산 합성 능력은 핵산 합성을 위한 산업적 공정을 창출하는데 활용될 수 있다. 이러한 산업적 공정에는 일반적으로 용액 또는 고체 지지체, 합성이 일어나는 특수 컨테이터 또는 용기(예를 들어 플로우 컬럼)에서 합성되는 핵산과 관련된 물질의 특정 구성, 효소 및 뉴클레오사이드 트리포스페이트를 추가하고 제거하기 위한 특정 기술(예를 들어 특수 전달 시스템 또는 미세유체공학 관련), 각 뉴클레오타이드 첨가 단계 후에 과도한 효소와 뉴클레오사이드 트리포스페이트를 제거하기 위한 특정 기술, 및 합성 후 반응 용기에서 효소를 제거하고 이를 고체 지지체, 완충제, 염 및 기타 용질과 같은 합성 중에 존재하는 물질로부터 분리하는 구체적인 방법이 포함된다.
핵산 합성을 위한 산업적 공정은 다양한 반응 온도, 예를 들어 섭씨 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 80, 90, 100, 110, 또는 120도 또는 그 사이의 온도에서 개발될 수 있다. 반응 온도는 일정할 수 있거나 반응 과정에서 임의의 방식으로, 예를 들어 시작 온도로부터 선형 또는 비선형 증가, 시작 온도로부터 선형 또는 비선형 감소, 주기적인 온도 변화 또는 이들의 조합에 의하여 어떤 방식으로든 변할 수 있다.
산업용 핵산 합성 공정에서는 각 뉴클레오타이드 추가 주기에 대해 예를 들어 주기당 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 또는 60초 또는 그 사이에 언제든지, 또는 주기당 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 또는 60분 또는 그 사이에 언제든지, 또는 주기당 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 또는 24시간 또는 그 사이의 언제든지의 서로 다른 반응 시간을 사용할 수 있다.
핵산 합성을 위한 산업적 공정은 다양한 규모로 설정되어 다양한 양의 핵산을 효율적으로 합성할 수 있다. 규모는 합성된 핵산의 fmol 양에서 몰 양 이상까지 다양할 수 있다. 예를 들어, 1x10-16, 2x10-16, 3x10-16, 4x10-16, 5x10-16, 6x10-16, 7x10-16, 8x10-16, 9x10-16, 1x10-15, 2x10-15, 3x10-15, 4x10-15, 5x10-15, 6x10-15, 7x10-15, 8x10-15, 9x10-15, 1x10-14, 2x10-14, 3x10-14, 4x10-14, 5x10-14, 6x10-14, 7x10-14, 8x10-14, 9x10-14, 1x10-13, 2x10-13, 3x10-13, 4x10-13, 5x10-13, 6x10-13, 7x10-13, 8x10-13, 9x10-13, 1x10-12, 2x10-12, 3x10-12, 4x10-12, 5x10-12, 6x10-12, 7x10-12, 8x10-12, 9x10-12, 1x10-11, 2x10-11, 3x10-11, 4x10-11, 5x10-11, 6x10-11, 7x10-11, 8x10-11, 9x10-11, 1x10-10, 2x10-10, 3x10-10, 4x10-10, 5x10-10, 6x10-10, 7x10-10, 8x10-10, 9x10-10, 1x10-9, 2x10-9, 3x10-9, 4x10-9, 5x10-9, 6x10-9, 7x10-9, 8x10-9, 9x10-9, 1x10-8, 2x10-8, 3x10-8, 4x10-8, 5x10-8, 6x10-8, 7x10-8, 8x10-8, 9x10-8, 1x10-7, 2x10-7, 3x10-7, 4x10-7, 5x10-7, 6x10-7, 7x10-7, 8x10-7, 9x10-7, 1x10-6, 2x10-6, 3x10-6, 4x10-6, 5x10-6, 6x10-6, 7x10-6, 8x10-6, 9x10-6, 1x10-5, 2x10-5, 3x10-5, 4x10-5, 5x10-5, 6x10-5, 7x10-5, 8x10-5, 9x10-5, 1x10-4, 2x10-4, 3x10-4, 4x10-4, 5x10-4, 6x10-4, 7x10-4, 8x10-4, 9x10-4, 1x10-3, 2x10-3, 3x10-3, 4x10-3, 5x10-3, 6x10-3, 7x10-3, 8x10-3, 9x10-3, 1x10-2, 2x10-2, 3x10-2, 4x10-2, 5x10-2, 6x10-2, 7x10-2, 8x10-2, 9x10-2, 1x10-1, 2x10-1, 3x10-1, 4x10-1, 5x10-1, 6x10-1, 7x10-1, 8x10-1, 9x10-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 80, 90 또는 100몰의 핵산 또는 그 사이의 모든 규모의 합성을 위해 특정 프로세스를 고안할 수 있다.
핵산 합성을 위한 산업적 공정은 임의의 구조의 뉴클레오타이드를 모든 핵산의 3' 말단에 추가하는데 필요한 모든 활성을 갖는 단일 효소에 의존할 수 있고, 또는 상기 공정은 특정 핵산에 특정 뉴클레오타이드를 첨가하는 것을 촉매하는 특수 효소에 의존할 수 있다. 예를 들어, 리보뉴클레오타이드를 추가하는데 사용되는 핵산 중합효소는 데옥시리보뉴클레오타이드를 추가하는데 사용되는 핵산 중합효소와 다를 수 있다. 다양한 염기 또는 변형을 포함하는 뉴클레오타이드를 추가하기 위해 다양한 핵산 중합효소를 사용할 수 있다. 다양한 핵산 중합효소를 사용하여 핵산의 3' 말단에 존재하는 서열 또는 핵산 내부에 존재하는 서열이 다른 핵산에 뉴클레오타이드를 추가할 수 있다. 다양한 핵산 중합효소를 사용하여 다양한 연결, 예를 들어 포스포로티오에이트 연결과 비교하여 표준 포스포디에스테르 연결을 갖는 뉴클레오타이드를 추가할 수 있다. 핵산의 다양한 서열 및/또는 구조를 합성하기 위하여, 산업 공정에서는 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 또는 1000개의 서로 다른 핵산 중합효소를 사용할 수도 있고 그 사이의 임의의 수를 사용할 수도 있다.
핵산 합성의 각 주기마다 핵산 중합효소가 첨가되어 이 주기에 필요한 특정 첨가 반응을 촉매한다. 핵산 중합효소는 단일 효소이거나 2개 이상의 효소의 혼합물일 수 있다.
효소적 올리고뉴클레오타이드 합성은 축퇴성 또는 혼합된 뉴클레오타이드를 올리고뉴클레오타이드의 특정 위치에 통합시킬 수 있다. 이는 특정 추가 주기에 대한 효소 추가 반응에 여러 뉴클레오사이드 트리포스페이트를 추가하는 것을 포함한다. 혼합 위치에 통합될 뉴클레오타이드의 구조에 따라 하나 이상의 핵산 중합효소가 추가되어 통합 반응을 촉매한다.
특정 위치에 축퇴성 또는 혼합된 뉴클레오타이드가 있는 핵산을 합성하는 경우, 특정 추가 주기에서 핵산의 단일 위치에 여러 뉴클레오타이드를 추가할 수 있도록 여러 효소를 추가할 수 있다.
축퇴 위치(degenerate position)에 통합된 뉴클레오타이드의 비율은 첨가 반응에 존재하는 각각의 뉴클레오사이드 트리포스페이트염의 농도, 효소 농도 및 다양한 효소의 상대적 비율에 영향을 미치는 반응 조건에 의해 영향을 받을 수 있다. 예를 들어, 2개 이상의 뉴클레오사이드 트리포스페이트의 혼합물 내 특정 뉴클레오사이드 트리포스페이트의 농도를 높이는 것은 전형적으로 해당 뉴클레오사이드 트리포스페이트의 통합 효율을 증가시킬 것이다. 유사하게, 혼합물 내 특정 뉴클레오사이드 트리포스페이트의 통합을 촉매하는 효소의 농도를 증가시키면 해당 뉴클레오사이드 트리포스페이트의 통합 빈도가 증가할 것이다. 이는 핵산 중합효소의 활성을 최적화하거나 혼합물에 존재하는 다른 핵산 중합효소에 비해 하나의 핵산 중합효소의 활성을 선호하도록 반응 조건(완충제, 염, 2가 양이온 및 반응 첨가제 또는 폴리에틸렌 글리콜, 폴리비닐피롤리돈, 글리세롤, 폴리아민, 세제, 소 혈청 알부민, DNA 결합 단백질 또는 포름아미드를 포함하지만 이에 국한되지 않는 안정화제의 존재; 완충제, 염, 2가 양이온, 뉴클레오사이드 트리포스페이트염 및 폴리에틸렌 글리콜, 폴리비닐피롤리돈, 글리세롤, 폴리아민, 세제, 소 혈청 알부민, DNA 결합 단백질 또는 포름아미드를 포함하지만 이에 국한되지 않는 기타 반응 성분; pH; 온도)을 변경함으로써 달성될 수 있다.
효소적으로 합성된 올리고뉴클레오타이드는 올리고튜클레오티드의 전체 길이 까지 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 또는 100000개 이상의 축퇴 뉴클레오타이드를 포함하여 임의의 수의 축퇴 뉴클레오타이드를 함유할 수 있다. 올리고뉴클레오타이드의 축퇴 위치는 4개의 표준 뉴클레오타이드 A, C, G 및 T 모두의 혼합물, 또는 염기의 하위 집합(예를 들어, A + C, A +G, A + T, C + G, C +T, G + T, A + C + G, A + C + T, A + G + T, C + G + T) 또는 표준 뉴클레오타이드와 모든 종류의 비천연 또는 변형된 뉴클레오타이드의 혼합물로 구성될 수 있다.
효소적 핵산 합성 공정에서, 합성되는 핵산은 용액 상태이거나 고체 지지체에 결합되거나 이들의 조합일 수 있다. 고체 지지체를 사용하는 경우, 핵산은 고체 지지체에 공유적으로 부착되거나 비공유적으로 부착될 수 있다.
합성 동안 핵산을 고정시키기 위해 다양한 고체 지지체가 사용될 수 있으며 이는 당업자에게 공지되어 있다. 여기에는 제어된 공극 유리(CPG) 비드, 아가로스 비드 또는 수지, 폴리스티렌 비드 또는 수지, PEG 비드 또는 수지, 실리카겔 비드 및 케미칼 그룹, 효소 또는 핵산의 고정화를 위해 개발된 기타 특수 재료이 포함되지만 이에 제한되지 않는다. 고체 지지체는 0.01~1000 마이크론 범위의 다양한 비드 크기와 0.01~1000 마이크론 범위의 기공 크기를 가질 수 있다.
효소적 핵산 합성 반응에 사용되는 핵산 중합효소는 용액 상태로 존재할 수도 있고, 아가로스 비드, 폴리스티렌 비드 또는 자기 비드를 포함하되 이에 국한되지 않는 고체 지지체 위에 고정될 수도 있다. 핵산 중합효소의 고정화는 고체 지지체에 대한 공유 결합을 통해 또는 고체 지지체와의 비공유 결합을 통해 발생할 수 있다. 핵산 중합효소를 고정하는데 사용되는 고체 지지체는 핵산 기질을 고정하는데 사용되는 것과 동일한 고체 지지체일 수도 있고, 다른 지지체일 수도 있다.
효소적 핵산 합성 반응(enzymatic nucleic acid synthesis reaction)에 사용되는 핵산 중합효소는 천연 기능에 기초하여 DNA 중합효소 또는 RNA 중합효소일 수 있다. DNA 중합효소의 경우, 중합효소는 패밀리 A, B, C, D, X, Y 및 RT를 포함하지만 이에 제한되지 않는 DNA 중합효소의 다른 공지된 패밀리 중 임의의 것에 속할 수 있다.
효소적 핵산 합성 반응에 사용되는 핵산 중합효소는 천연 효소(natural enzyme)이거나 새로운 핵산 합성에 대한 유용성을 높이기 위해 사람의 손에 의해 서열이나 구조가 변경되었음을 의미하는 가공된 효소일 수 있다.
본 개시내용은 핵산 분자의 3' 말단에 단일 뉴클레오타이드를 추가할 수 있는 7개의 신규 핵산 중합효소를 기술한다. 이들 효소의 서열 번호는 아래 표 1에 제시되어 있으며, 이들의 활성은 실시예 1에 기재되어 있다.
표 1: 핵산 중합효소 | SEQ ID NOs: | ||||||
Enzyme name | Accession number | Plasmid name | Species | A | B | C | D |
EDS017 | Q04049 | PP1077 | Saccharomyces cerevisiae | 1 | 11 | 21 | 31 |
EDS024 | BAD02935 | PP1084 | Takifugu rubripes | 2 | 12 | 22 | 32 |
EDS029 | KTA96827.1 | PP1089 | Candida glabrata | 3 | 13 | 23 | 33 |
EDS030 | XP_011273936.1 | PP1090 | Wickerhamomyces ciferrii | 4 | 14 | 24 | 34 |
EDS053 | AYW42506.1 | PP1113 | Pseudomonas aeruginosa | 5 | 15 | 25 | 35 |
EDS054 | WP_124690524.1 | PP1114 | Pigmentiphaga sp. H8 | 6 | 16 | 26 | 36 |
EDS066 | XP_031753771.1 | PP1126 | Xenopus tropicalis | 7 | 17 | 27 | 37 |
EDS082 | XP_011273936.1 | PP1142 | Wickerhamomyces ciferrii | 8 | 18 | 28 | 38 |
EDS048 | DAA14763.1 | PP1108 | Bos taurus | 9 | 19 | 29 | 39 |
EDS015 | NP_001036693 | PP1075 | Mus musculus | 10 | 20 | 30 | 40 |
여기서 A 열의 SEQ ID NO는 천연 서열(아미노산)이다.
B 열의 SEQ ID NO는 복제된 유전자 서열(핵산)이다.
C 열의 SEQ ID NO는 발현된 단백질 서열(아미노산)이다.
D열의 서열 번호는 발현 플라스미드 서열(핵산)이다.
위에서 언급한 바와 같이, 핵산 중합효소는 핵산 기질의 3' 말단에 단일 뉴클레오타이드를 추가하는 부분적인 능력을 가질 수 있으며, 이는 반응 중에 핵산 기질에 단일 뉴클레오타이드를 추가하는 효율이 100% 이하일 수 있음을 의미한다. 이러한 효율성을 높이기 위해 핵산 중합효소를 더욱 효율적으로 조작할 수 있다. 이는 모체 효소(parental enzyme)보다 반응에서 더 높은 첨가 효율을 갖는 원래 효소의 변종이 생성된다는 것을 의미한다. 핵산 중합효소는 기질 특이성을 변경하도록 조작될 수도 있다. 예를 들어, T로 끝나는 핵산의 3' 말단에 뉴클레오타이드를 효율적으로 추가하는 핵산 중합효소는 임의의 뉴클레오타이드로 끝나는 핵산에 뉴클레오타이드를 효율적으로 추가하도록 조작될 수 있다. 또 다른 예로서, A를 핵산의 3' 말단에 효율적으로 첨가하는 핵산 중합효소는 더 넓은 기질 특이성을 위해 조작될 수 있고, 따라서 변이체 효소(variant enzyme)는 핵산 분자의 3' 말단에 임의의 뉴클레오타이드를 효율적으로 첨가할 수 있다. 또 다른 예에서, 처리적 방식으로 반응에서 핵산의 3' 말단에 다중 뉴클레오타이드를 추가하는 핵산 중합효소는 반응 동안 3' 말단에 단일 뉴클레오타이드만 추가하도록 조작될 수 있다. 추가 예에서, 데옥시리보스 뉴클레오타이드를 핵산의 3' 말단에 효율적으로 첨가하는 핵산 중합효소는 리보뉴클레오타이드를 효율적으로 첨가하도록 조작될 수 있다. 추가 예에서, DNA 분자의 3' 말단에 데옥시리보스 뉴클레오타이드를 효율적으로 첨가하는 핵산 중합효소는 데옥시리보뉴클레오타이드를 RNA 분자에 효율적으로 첨가하도록 조작될 수 있다. 마지막 예에서, DNA 분자의 3' 말단에 리보뉴클레오타이드를 효율적으로 추가하는 핵산 중합효소는 RNA 분자의 3' 말단에 리보뉴클레오타이드를 효율적으로 추가하도록 조작될 수 있다. 이러한 예는 완전한 것이 아니며, 실제로 이러한 활성이 결여되어 있거나 낮은 효율로 이러한 활성을 나타내는 출발 효소를 조작함으로써 임의의 특정한 바람직한 핵산 중합효소 활성을 조작하는 것이 가능하다.
다음 리뷰 기사에 나열된 것을 포함하되 이에 국한되지 않는 단백질 공학을 위한 많은 접근 방식과 방법이 문헌에 설명되어 있다: Leatherbarrow 1986, Zoller 1991, Lutz 2000, Leisola 2007, Eisenbeis 2010, O'Fagain 2011, Foo 2012, Zawaira 2012, Marcheschi 2013, Woodley 2013, Johnson 2014, Packer 2015, Shin 2015, Chen 2016, Kaushik 2016, Swint-Kruse 2016, Wrenbeck 2017, Bornscheuer 2018, Lutz 2018, Singh 2018, Sinha 2019, Wilding 2019, Yang 2019.
일반적으로 단백질 공학은 관심 효소를 코딩하는 유전자 서열을 다양화하기 위해 하나 이상의 방법을 사용하고, 이어서 하나 이상의 관심 품질이 개선된 변이 효소를 코딩하는 유전자를 선택하는데 사용되는 하나 이상의 선택 또는 스크리닝 방법을 사용한다. 관심 품질에는 다음이 포함되지만 이에 제한되지는 않는다: 특정 반응 조건에서 또는 특정 기질을 변형할 때 뉴클레오타이드 추가 효율(nucleotide addition efficiency); 핵산 기질과 관련된 기질 특이성(substrate specificity); 억제제에 대한 내성(resistance); 뉴클레오사이드 트리포스페이트와 관련된 기질 특이성; 고온에 노출되었을 때의 안정성; 염, 피로인산염 또는 기타 반응 생성물, 또는 기타 화학물질 또는 화합물의 반응에서의 존재와 같은 모 효소를 비활성화할 수 있는 조건 하에서의 안정성; 전술한 것 중 어느 것의 반응에서 고농도; 또는 효소적 핵산 합성 과정에 대한 적합성을 향상시킬 수 있는 효소의 다른 품질.
관심 핵산 중합효소를 코딩하는 유전자를 다양화하는 방법에는 다음이 포함되나 이에 국한되지는 않는다: 점 돌연변이의 도입을 의미하는 돌연변이 유발; 효소 코딩 서열 내에서 다양한 길이의 삽입 및 결실(insertions and deletion)의 도입; 코딩 서열의 5' 또는 3' 말단에서 다른 서열과의 융합; 다형성의 재분류를 초래하는 관련 코딩 서열과의 상동 서열 교환; 및 서열 다양성을 생성하는 다른 수단.
주형 독립적 핵산 중합효소의 하위 집합에는 핵산 중합효소 활성에 필수적이지 않고 DNA 합성 또는 복구에 관여하는 다른 단백질과의 상호작용을 중재할 수 있는 BRCT 도메인이 포함되어 있다(Callebaut 1997, Repasky 2004). BRCT 도메인을 제거하기 위한 단백질의 절단은 말단 데옥시뉴클레오티딜트랜스퍼라제에서 DNA 중합효소 활성을 자극하는 것으로 보고되었다(Mueller 2009). BRCT 도메인을 제거하는 유사한 표적 절단을 사용하여 다른 TINAP의 활동을 변경할 수 있다.
하나 이상의 관심 품질이 개선된 효소를 코딩하는 유전자를 선택하는데 사용되는 방법 및 접근법에는 소량으로 많은 수의 효소 변이체를 효율적으로 처리할 수 있는 미세액적 또는 에멀젼의 시험관내 구획화를 사용하는 접근법이 포함된다. 러한 접근법은 일반적인 방식으로 그리고 핵산 처리 효소에 대한 특정 적용으로 문헌에 설명되어 있다(Tawfik 1998, Ghadessy 2001, Diehl 2006, Griffiths 2006, Miller 2006, Ghadessy 2007, Tay 2010, Takeuchi 2014).
실시예
실시예 1: 용액 내 올리고뉴클레오타이드에 대한 단일 뉴클레오타이드 첨가
DNA 중합효소, 효소 발현 및 정제:
N-말단에 6-히스티딘 태그를 각각 포함하는 표 1에 나열된 DNA 중합효소를 코딩하는 유전자(서열 번호: 21-30)는 상업적인 방법으로 유전자 합성 공급 업체에 의해서 합성되고 E. coli에서 높은 카피수를 부여하는 MB1 플라스미드 레플리콘을 갖는 박테리아 발현 플라스미드에 클로닝된 핵산 서열(서열 번호: 11-20)로 설계되었다. 상기 플라스미드의 DNA 중합효소 유전자 삽입 부위에는 각 중합효소의 아라비노스 유도성 발현이 가능하도록 라비노스 유도성 프로모터와 람다 T1 터미네이터 옆에 있다(flanked). 클로닝 후 발현 구조를 서열 검증한다. 본 개시내용에서 다루는 DNA 중합효소에 대한 발현 구조물의 전체 서열은 서열 번호 31-40에 제시되어 있다.
EDS082를 코딩하는 유전자의 코딩 서열은 EDS030을 코딩하는 서열을 절단하여 얻었다. EDS030의 N-말단에 존재하는 BRCT 도메인을 코딩하는 서열은 다른 중합효소에 대해 기술된 바와 같이 제거되었으며(Mueller 2009), 단축된 코딩 서열의 시작 부분에 메티오닌 코돈이 삽입되었다.
발현 플라스미드는 E. coli 균주 BL21로 형질전환되고 단일 콜로니는 배양 및 단백질 발현을 위해 선택된다. 박테리아 세포를 37℃의 LB 배지에서 성장시켜 log phase 배양하고 L-아라비노스를 첨가하여 유도한다. 15℃에서 18시간 동안 배양한 후 원심분리에 의해 배양물을 수확하고 수집된 E. coli 세포를 용해한다. DNA 중합효소는 제조업체의 지침에 따라 니켈 친화성 크로마토그래피로 정제된다. DNA 중합효소를 Millipore(Darmstadt, Germany)에서 판매하는 AMICON® Ultra-centrifugal filter로 농축된 이미다졸 용액 으로 용출시키고, 50mM KPO4, pH7.3, 100mM NaCl, 1.43mM 베타 머캅토에탄올, 0.05% Triton-X100 및 50% 글리세롤로 구성된 저장 완충액으로 변경되었다.
올리고뉴클레오타이드 및 dNTP 풀을 사용한 시험관 내 뉴클레오타이드 추가 분석
효소 활성은 pH 7.5에서 50mM 칼륨 아세테이트와 20mM 트리스 아세테이트로 구성된 완충액에서 반응을 수행하여 분석된다. 반응 완충액에는 10mM 마그네슘 아세테이트와 250μM 염화코발트가 첨가된다. 반응은 500μM dNTP, 10μM의 단일 가닥 DNA 올리고뉴클레오타이드 및 1μg의 효소/10μl 반응의 존재 하에 수행된다. 15℃에서 시작하여 1℃/분의 속도로 50℃까지 올라가는 온도 구배를 사용하여 반응을 인큐베이션한다. 반응은 10 μl 부피로 수행되고 얼음 위에 셋업된다.
활성 스크리닝을 위해 단일 가닥 DNA 올리고뉴클레오타이드의 등몰 혼합물이 사용된다: PG5861(GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5859(GTCCTCAATCGCACTGGAAG, 서열 번호 43); PG5860(GTCCTCAATCGCACTGGAAC, 서열 번호 44); PG5858(GTCCTCAATCGCACTGGAAA, 서열 번호 42). 단일 가닥 올리고뉴클레오타이드의 혼합물은 dATP, dTTP, dGTP 및 dCTP의 등몰 혼합물과 결합된다. 올리고뉴클레오타이드는 Eurofins Genomics(켄터키주 루이빌)에서 합성하고 dNTP는 New England Biolabs(메사추세츠주 베벌리)에서 구입하였다.
동일한 부피의 2x NOVEXTM TBE-Urea 샘플 완충액(ThermoFisher, Waltham, MA)을 첨가하고 70℃에서 3분 동안 가열하여 반응을 중단시킨다. 샘플을 냉각시키고 15μl를 NOVEXTM TBE-Urea 폴리아크릴아미드 겔(15%, ThermoFisher, Waltham, MA)에 첨가하고, 150V에서 전기영동하고, 메틸렌 블루로 염색하고, 탈이온수로 탈염색(destain)하고, AZURETM 200 젤 이미징 워크스테이션(Azure Biosystems, Dublin, CA)을 사용하여 백색광으로 이미지화한다.
10가지 DNA 중합효소의 활성 평가 예가 도 2에 나와 있다. 다양한 효소는 단일 가닥 올리고뉴클레오타이드에 하나 또는 여러 개의 뉴클레오타이드를 추가하는 경향을 나타내며, 이는 효소 핵산 합성 공정에 대한 적합성을 나타낼 수 있다.
겔 전기영동에 의한 단일 뉴클레오타이드 첨가 분석
개별 dNTP를 사용한 효소 활성은 pH 7.5에서 50mM 칼륨 아세테이트와 20mM 트리스 아세테이트로 구성된 완충액에서 반응을 수행하여 분석된다. 반응 완충액에는 10mM 마그네슘 아세테이트와 250μM 염화코발트가 첨가된다. 반응은 500μM dNTP, 10μM의 단일 가닥 DNA 올리고뉴클레오타이드 및 1μg의 효소/10μl 반응의 존재 하에 수행된다. 반응물을 30℃에서 15분 동안 인큐베이션한다. 반응은 10 μl 부피로 수행되었으며 얼음 위에 셋업되었다.
각 반응에는 다음과 같은 개별 dNTP 및 DNA 올리고뉴클레오타이드 쌍이 사용된다: dTTP + PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); dGTP + PG5864 (GTCCTCAATCGCACTGGAATT, 서열 번호 46); dATP + PG5865 (GTCCTCAATCGCACTGGAATTG, 서열 번호 47); dCTP + PG5866 (GTCCTCAATCGCACTGGAATTGA, 서열 번호 48). 표준 올리고뉴클레오타이드도 분석에 사용된다: PG5867 (GTCCTCAATCGCACTGGAATTGAC, 서열 번호 54).
동일한 부피의 2x NOVEXTM TBE-Urea 샘플 완충액(ThermoFisher, Waltham, MA)을 첨가하고 70℃에서 3분 동안 가열하여 반응을 중단시킨다. 샘플을 냉각시키고 15μl를 NOVEXTM TBE-Urea 폴리아크릴아미드 겔(15%, ThermoFisher, Waltham, MA)에 첨가하고, 150V에서 전기영동하고, 메틸렌 블루로 염색하고, 탈이온수로 탈염색(destain)하고, AZURETM 200 젤 이미징 워크스테이션(Azure Biosystems, Dublin, CA)을 사용하여 백색광으로 이미지화한다.
도 3A는 위에 나열된 4개의 서로 다른 올리고뉴클레오타이드 기질에 단일 뉴클레오타이드를 효율적으로 첨가하는 것을 보여준다.
순차적인 뉴클레오타이드 첨가에 대한 분석
순차적인 뉴클레오타이드 첨가 반응(Sequential nucleotide addition reaction)은 pH 7.5에서 50mM 칼륨 아세테이트와 20mM 트리스 아세테이트로 구성된 완충액에서 수행된다. 반응 완충액에는 10mM 마그네슘 아세테이트와 250μM 염화코발트가 첨가된다. 반응은 500μM dNTP, 10μM의 단일 가닥 DNA 올리고뉴클레오타이드 및 1μg의 효소/10μl 반응의 존재 하에 수행된다. 반응물을 30℃에서 15분 동안 인큐베이션된다. 여러 dNTP를 추가하기 위한 순차적 반응을 수행할 때 반응 용량은 100ul까지 확장된다. 초기 반응은 다음 서열 PG5861(GTCCTCAATCGCACTGGAAT, 서열 번호 45) 및 뉴클레오사이드 트리포스페이트로서 dTTP를 갖는 단일 가닥 DNA 올리고뉴클레오타이드를 사용하여 수행된다.
100℃에서 3분 동안 끓여서 반응을 중단시키고 제조업체의 지침에 따라 Zymo Research(Irvine, CA)의 Oligo뉴클레오타이드 Clean and Concentrator 키트를 사용하여 실리카 컬럼의 반응 성분으로부터 올리고뉴클레오타이드를 정제하고 증류수에서 용출했다. 정제된 올리고뉴클레오타이드의 농도는 Thermo Scientific(Waltham, MA)의 NANODROPTM One 분광광도계를 사용하여 측정하고, 겔 전기영동을 위해 따로 보관해 둔 분취량을 사용한다. 남은 정제된 올리고뉴클레오타이드는 출발 올리고뉴클레오타이드와 동일한 과정에서 dGTP를 사용하는 추가 반응에 사용된다.
다음 올리고뉴클레오타이드는 샘플에 추가하고 중복 분석을 실행하여 표준으로 사용된다(그림 4B, D, F 및 H 참조): PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5864 (GTCCTCAATCGCACTGGAATT, 서열 번호 46); PG5865 (GTCCTCAATCGCACTGGAATTG, 서열 번호 47); PG5866 (GTCCTCAATCGCACTGGAATTGA, 서열 번호 48); and PG5867 (GTCCTCAATCGCACTGGAATTGAC, 서열 번호 54).
겔 전기영동에 의한 분석을 위해, 동일한 부피의 2x NOVEXTM TBE-Urea 샘플 완충액(ThermoFisher, Waltham, MA)을 첨가하여 샘플을 희석하고 70℃에서 3분간 가열한다. 샘플을 냉각시키고 15μl를 NOVEXTM TBE-Urea 폴리아크릴아미드 겔(15%, ThermoFisher, Waltham, MA)에 첨가하고, 150V에서 전기영동하고, 메틸렌 블루로 염색하고, 탈이온수로 탈염색하고, AZURETM 200 젤 이미징 워크스테이션(Azure Biosystems, Dublin, CA)을 사용하여 백색광으로 이미지화한다.
도 3B는 서열 번호 45의 주어진 서열을 갖는 올리고뉴클레오타이드 기질에 2개의 뉴클레오타이드를 효율적으로 순차적으로 첨가하는 것을 보여준다.
모세관 전기영동에 의한 단일 뉴클레오타이드 첨가 분석
개별 dNTP 올리고뉴클레오타이드 쌍을 사용하는 효소 활성은 pH 7.5에서 50mM 칼륨 아세테이트와 20mM 트리스 아세테이트로 구성된 완충액에서 반응을 수행하여 분석된다. 응 완충액에는 10mM 마그네슘 아세테이트와 250μM 염화코발트가 첨가된다. 반응은 500μM dNTP, 10μM의 단일 가닥 DNA 올리고뉴클레오타이드 및 1μg의 효소/10μl 반응의 존재 하에 수행된다. 반응물을 30℃에서 15분 동안 인큐베이션한다. 반응은 10 μl 부피로 수행되고 얼음 위에 설정된다.
사용된 올리고뉴클레오타이드: PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5864 (GTCCTCAATCGCACTGGAATT, 서열 번호 46); PG5872 (GTCCTCAATCGCACTGGAATG, 서열 번호 53); PG5859 (GTCCTCAATCGCACTGGAAG, 서열 번호 43); PG5868 (GTCCTCAATCGCACTGGAAGT, 서열 번호 49); PG5869 (GTCCTCAATCGCACTGGAAGC, 서열 번호 50); PG5858 (GTCCTCAATCGCACTGGAAA, 서열 번호 42).
각 올리고뉴클레오타이드에 대한 효소 첨가는 개별 반응에서 dATP, dTTP, dGTP 및 dCTP를 사용하여 개별적으로 평가된다. 100℃에서 3분 동안 끓여서 반응을 중단시키고 올리고뉴클레오타이드를 제조업체의 지침에 따라 Zymo Research(Irvine, CA)의 Oligo뉴클레오타이드 Clean and Concentrator 키트를 사용하여 실리카 컬럼의 반응 성분으로부터 정제하고 증류수에서 용리시켰다. 그런 다음 정제된 올리고뉴클레오타이드를 24-모세관 어레이를 사용하여 Agilent Technologies(Santa Clara, CA)의 Agilent Oligo Pro II 모세관 전기영동 시스템에서 분석한다. 10초 동안 9-12 kV 범위의 주입 방법을 사용하여 분석하기 위해 에 정제된 올리고뉴클레오타이드를 ~0.5-2 μM로 희석하고, 15 kV에서 70분 동안 분리한다. 데이터는 Agilent Oligo Pro II 데이터 분석 소프트웨어 2.0.0.3(Agilent Technologies, Santa Clara, CA)을 사용하여 분석된다. 반응 분석은 각 샘플에 대해 두 번의 독립적인 실행을 통해 수행된다. 한 번의 실행에는 시작 올리고뉴클레오타이드의 순도와 전환율을 평가하기 위해 Agilent Oligo Pro II에 순수 샘플만 포함되어 있다(도 4A, 4C, 4E 및 4G). 두 번째 실행은 반응을 수행한 후 정제된 올리고뉴클레오타이드의 크기를 정확하게 측정하기 위해 각 샘플에 첨가된(spiked) 표준물질을 포함하여 수행된다(도 4B, 4D, 4F 및 4H).
다음 올리고뉴클레오타이드 표준은 ~1μM 최종 농도로 첨가된다: PG1350 (GCGTCACGCTACCAACCA, 서열 번호 41); PG5870 (GTCCTCAATCGCACTGGAAACATCAAGGTC, 서열 번호 51); PG5871 (GTCCTCAATCGCACTGGAAACATCAAGGTCATACGGAACG, 서열 번호 52). 각 특정 반응에 사용되는 올리고뉴클레오타이드도 표준 물질과 함께 ~1μM로 첨가된다.
Agilent Oligo Pro II 기기에서 실행된 대표적인 모세관 전기영동의 프로필이 도 4A-H에 나와 있다. 도 4A 및 4B는 효소 반응에서 처리되지 않은 대조 올리고뉴클레오타이드의 모세관 전기영동 실행을 보여준다. 도 4C 및 4D는 올리고뉴클레오타이드 PG5861(서열 번호 45)과 dTTP 및 효소 EDS082(표 1 참조)의 반응 후 단일 가닥 올리고뉴클레오타이드에 단일 뉴클레오타이드의 부분 첨가를 보여준다. 도 4E 및 4F는 올리고뉴클레오타이드 PG5861(서열 번호 45)과 dTTP 및 효소 EDS054(표 1 참조)의 반응 후 단일 가닥 올리고뉴클레오타이드에 단일 뉴클레오타이드의 효율적인 첨가를 보여준다. 도 4G 및 4H는 올리고뉴클레오타이드 PG5861(서열 번호 45)과 dTTP 및 효소 EDS066(표 1 참조)의 반응 후 단일 가닥 올리고뉴클레오타이드에 1, 2, 3, 4 및 5개의 뉴클레오타이드를 첨가하는 것을 보여준다.
단일 뉴클레오타이드 첨가를 보여주는 50가지 대표적인 반응의 결과는 아래 표 2에 요약되어 있다.
N은 이들 반응에서 기질 역할을 하는 올리고뉴클레오타이드의 뉴클레오타이드 길이를 의미한다.
% <N은 N보다 짧은 생성물(예를 들어 올리고뉴클레오타이드 기질의 분해 생성물)의 백분율을 의미한다.
% N은 N의 길이를 갖는 생성물(예를 들어 미반응 올리고뉴클레오타이드 기질)의 백분율을 의미한다.
% N+1은 N보다 하나의 뉴클레오타이드가 더 긴 생성물(예를 들어 원하는 연장 생성물)의 비율을 의미한다.
% N+>1은 N보다 2개 이상의 뉴클레오타이드가 더 긴 생성물(예를 들어 2개 이상의 추가된 뉴클레오타이드를 수용한 올리고뉴클레오타이드 기질의 연장 생성물)의 백분율을 의미한다.
표는 각 실시예에서 원하는 N+1 확장 생성물의 수율을 명확하게 보여주며, 단일 뉴클레오타이드 추가 효율은 36%~100% 범위이다.
Table 2: 50가지 대표적인 첨가반응 결과 | ||||||||
Reaction # | Enzyme used | Substrate (SEQ ID NO) | dNTP | % <N | % N | % N+1 | % N+>1 | Total |
29 | EDS030 | 45 | G | 0% | 0% | 100% | 0% | 100% |
30 | EDS053 | 45 | G | 0% | 0% | 100% | 0% | 100% |
31 | EDS054 | 45 | G | 0% | 0% | 100% | 0% | 100% |
389 | EDS030 | 53 | A | 0% | 5% | 95% | 0% | 100% |
393 | EDS082 | 53 | A | 0% | 6% | 94% | 0% | 100% |
388 | EDS029 | 53 | A | 0% | 6% | 94% | 0% | 100% |
392 | EDS066 | 53 | A | 0% | 6% | 94% | 0% | 100% |
390 | EDS053 | 53 | A | 5% | 6% | 90% | 0% | 100% |
391 | EDS054 | 53 | A | 0% | 4% | 89% | 7% | 100% |
236 | EDS066 | 43 | C | 0% | 9% | 84% | 8% | 100% |
387 | EDS017 | 53 | A | 0% | 0% | 80% | 20% | 100% |
211 | EDS054 | 43 | T | 0% | 20% | 80% | 0% | 100% |
77 | EDS030 | 46 | G | 0% | 12% | 67% | 21% | 100% |
78 | EDS053 | 46 | G | 0% | 21% | 66% | 13% | 100% |
230 | EDS017 | 43 | C | 0% | 0% | 66% | 34% | 100% |
19 | EDS054 | 45 | T | 14% | 21% | 65% | 0% | 100% |
208 | EDS029 | 43 | T | 0% | 0% | 65% | 35% | 100% |
400 | EDS029 | 53 | T | 0% | 0% | 65% | 35% | 100% |
27 | EDS017 | 45 | G | 0% | 0% | 65% | 35% | 100% |
326 | EDS017 | 42 | C | 0% | 18% | 65% | 18% | 100% |
220 | EDS029 | 43 | G | 0% | 0% | 64% | 36% | 100% |
81 | EDS082 | 46 | G | 6% | 30% | 60% | 4% | 100% |
279 | EDS017 | 49 | C | 0% | 40% | 60% | 0% | 100% |
242 | EDS017 | 49 | A | 0% | 0% | 59% | 41% | 100% |
90 | EDS053 | 46 | C | 0% | 0% | 59% | 41% | 100% |
207 | EDS017 | 43 | T | 0% | 35% | 58% | 7% | 100% |
219 | EDS017 | 43 | G | 0% | 18% | 58% | 24% | 100% |
79 | EDS054 | 46 | G | 0% | 9% | 53% | 38% | 100% |
18 | EDS053 | 45 | T | 25% | 22% | 52% | 0% | 100% |
87 | EDS017 | 46 | C | 7% | 31% | 48% | 14% | 100% |
62 | EDS017 | 46 | T | 0% | 41% | 48% | 11% | 100% |
440 | EDS066 | 50 | A | 9% | 38% | 48% | 4% | 100% |
463 | EDS054 | 50 | G | 18% | 35% | 47% | 0% | 100% |
14 | EDS017 | 45 | T | 0% | 0% | 47% | 53% | 100% |
422 | EDS017 | 53 | C | 0% | 33% | 46% | 21% | 100% |
33 | EDS082 | 45 | G | 14% | 41% | 45% | 0% | 100% |
460 | EDS029 | 50 | G | 5% | 43% | 44% | 8% | 100% |
91 | EDS054 | 46 | C | 18% | 11% | 43% | 29% | 100% |
403 | EDS054 | 53 | T | 9% | 49% | 42% | 0% | 100% |
316 | EDS029 | 42 | G | 0% | 11% | 42% | 47% | 100% |
17 | EDS030 | 45 | T | 18% | 41% | 42% | 0% | 100% |
232 | EDS029 | 43 | C | 0% | 0% | 41% | 59% | 100% |
64 | EDS029 | 46 | T | 0% | 0% | 40% | 60% | 100% |
200 | EDS066 | 43 | A | 0% | 60% | 40% | 0% | 100% |
332 | EDS066 | 42 | C | 12% | 48% | 40% | 0% | 100% |
199 | EDS054 | 43 | A | 0% | 60% | 40% | 0% | 100% |
21 | EDS082 | 45 | T | 10% | 52% | 37% | 0% | 100% |
212 | EDS066 | 43 | T | 0% | 14% | 37% | 48% | 100% |
15 | EDS017 | 45 | T | 10% | 53% | 37% | 0% | 100% |
195 | EDS017 | 43 | A | 0% | 64% | 36% | 0% | 100% |
리보뉴클레오타이드 첨가 분석
4개의 NTP의 동일한 몰 혼합을 사용하는 효소 활성은 pH 7.5에서 50mM 칼륨 아세테이트와 20mM 트리스 아세테이트로 구성된 완충액에서 반응을 수행하여 분석된다. 반응 완충액에는 10mM 마그네슘 아세테이트와 250μM 염화코발트가 첨가된다. 반응은 500μM NTP, 10μM의 단일 가닥 DNA 올리고뉴클레오타이드 및 1μg의 효소/10μl 반응의 존재 하에 수행된다. 반응은 15℃에서 시작하여 1℃/분의 속도로 37℃까지 올라가는 다양한 온도에서 인큐베이션된다. 반응은 10 μl 부피로 수행되고 얼음 위에 셋업된다.
초기 활성 스크리닝(그림 5A)을 위해 단일 가닥 DNA 올리고뉴클레오타이드의 등몰 혼합물이 사용된다: PG5861 (GTCCTCAATCGCACTGGAAT, 서열 번호 45); PG5859 (GTCCTCAATCGCACTGGAAG, 서열 번호 43); PG5860 (GTCCTCAATCGCACTGGAAC, 서열 번호 44); PG5858 (GTCCTCAATCGCACTGGAAA, 서열 번호 42). 단일 가닥 DNA 올리고뉴클레오타이드(도 5B)에 NTP의 첨가를 분석하기 위해, PG5861(GTCCTCAATCGCACTGGAAT, 서열 번호 45)이 각 반응에 사용된다.
동일한 부피의 2x NOVEXTM TBE-Urea 샘플 완충액(ThermoFisher, Waltham, MA)을 첨가하고, 3분 동안 70℃로 가열하여 반응을 중지 한다. 샘플을 냉각시키고 15μl를 NOVEXTM TBE-Urea 폴리아크릴아미드 겔(15%, ThermoFisher, Waltham, MA)에 첨가하고, 150V에서 전기영동하고, 메틸렌 블루로 염색하고, 물로 탈염색하고, AZURETM 200 겔 이미징 워크스테이션을 사용하여 백색광으로 이미지화한다.
DNA 올리고뉴클레오타이드에 리보뉴클레오타이드를 첨가한 결과의 예를 도 5에 나타내었다. 효소 EDS017, EDS024, EDS029, EDS030, EDS066, EDS082, EDS048 및 EDS015는 모두 리보뉴클레오타이드를 통합하는 능력을 보여주었다. 대부분의 경우 이러한 통합은 1-3개의 뉴클레오타이드로 제한되었다.
DNA 올리고뉴클레오타이드의 말단에 리보뉴클레오타이드를 첨가하는 다양한 효소의 능력이 표 3에 요약되어 있다.
Table 3: DNA 중합효소에 의한 DNA 올리고뉴클레오타이드에 대한 리보뉴클레오타이드 첨가 요약 | |
Enzyme | 추가된 리보뉴클레오타이드의 최대 개수 |
EDS017 | 2 |
EDS024 | 2 |
EDS029 | 1 |
EDS030 | 10 |
EDS053 | 0 |
EDS054 | 0 |
EDS066 | 2 |
EDS082 | 4 |
EDS048 | 3 |
EDS015 | 2 |
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Leu Glu Arg Ile Pro Arg Asp Glu Cys Thr Leu His Asn Glu Phe Met
290 295 300
Ser Asp Leu Val Ser Gln Ile Asp Pro Leu Val Gln Phe Thr Ile Gly
305 310 315 320
Gly Ser Tyr Arg Arg Gly Ser Pro Thr Cys Gly Asp Val Asp Phe Ile
325 330 335
Ile Thr Lys Pro Asn Ala Asp Asn Glu Glu Met Lys Glu Ile Leu Glu
340 345 350
Lys Ile Leu Val Lys Ile Glu Gln Val Gly Tyr Leu Lys Cys Ser Leu
355 360 365
Gln Lys Lys His Ser Thr Lys Phe Leu Ser Gly Cys Ala Leu Pro Pro
370 375 380
Asn Tyr Ala Ser Arg Leu Pro Glu Tyr Ser Glu Gly Lys Trp Gly Lys
385 390 395 400
Cys Arg Arg Ile Asp Phe Leu Met Val Pro Trp Lys Glu Arg Gly Ala
405 410 415
Ala Phe Ile Tyr Phe Thr Gly Asn Asp Tyr Phe Asn Arg Leu Ile Arg
420 425 430
Leu Lys Ala Val Lys Asn Gly Leu Val Leu Asn Glu Ser Gly Leu Phe
435 440 445
Lys Arg Ile Lys Tyr Val Gln Gly Lys Asn Val Glu Asp Lys Thr Met
450 455 460
Leu Ile Glu Ser Phe Ser Glu Lys Lys Ile Phe Lys Leu Leu Gly Phe
465 470 475 480
Lys Tyr Val Pro Pro Glu Gln Arg Asn Phe Gly Ala Asn Asn Pro Pro
485 490 495
Ser Lys Leu Gly Lys His Leu Asp Gln Phe Arg Ile Asp His Lys Tyr
500 505 510
Phe Asp Lys Val Val Lys Glu Glu Ile Ile Asp Asp Asp Val Ile Glu
515 520 525
Val Asp
530
<210> 5
<211> 349
<212> PRT
<213> Pseudomonas aeruginosa
<400> 5
Met Arg Lys Ile Ile His Ile Asp Cys Asp Cys Phe Tyr Ala Ala Leu
1 5 10 15
Glu Met Arg Asp Asp Pro Ser Leu Arg Gly Lys Ala Leu Ala Val Gly
20 25 30
Gly Ser Pro Asp Lys Arg Gly Val Val Ala Thr Cys Ser Tyr Glu Ala
35 40 45
Arg Ala Tyr Gly Val Arg Ser Ala Met Ala Met Arg Thr Ala Leu Lys
50 55 60
Leu Cys Pro Asp Leu Leu Val Val Arg Pro Arg Phe Asp Val Tyr Arg
65 70 75 80
Ala Val Ser Lys Gln Ile His Ala Ile Phe Arg Asp Tyr Thr Asp Leu
85 90 95
Ile Glu Pro Leu Ser Leu Asp Glu Ala Tyr Leu Asp Val Ser Ala Ser
100 105 110
Pro His Phe Ala Gly Ser Ala Thr Arg Ile Ala Gln Asp Ile Arg Arg
115 120 125
Arg Val Ala Glu Glu Leu Arg Ile Thr Val Ser Ala Gly Val Ala Pro
130 135 140
Asn Lys Phe Leu Ala Lys Ile Ala Ser Asp Trp Arg Lys Pro Asp Gly
145 150 155 160
Leu Phe Val Ile Thr Pro Glu Gln Val Asp Gly Phe Val Ala Glu Leu
165 170 175
Pro Val Ala Lys Leu His Gly Val Gly Lys Val Thr Ala Glu Arg Leu
180 185 190
Ala Arg Met Gly Ile Arg Thr Cys Ala Asp Leu Arg Gln Gly Ser Lys
195 200 205
Leu Ser Leu Val Arg Glu Phe Gly Ser Phe Gly Glu Arg Leu Trp Gly
210 215 220
Leu Ala His Gly Ile Asp Glu Arg Pro Val Glu Val Asp Ser Arg Arg
225 230 235 240
Gln Ser Val Ser Val Glu Cys Thr Phe Asp Arg Asp Leu Pro Asp Leu
245 250 255
Ala Ala Cys Leu Glu Glu Leu Pro Thr Leu Leu Glu Glu Leu Asp Gly
260 265 270
Arg Leu Gln Arg Leu Asp Gly Ser Tyr Arg Pro Asp Lys Pro Phe Val
275 280 285
Lys Leu Lys Phe His Asp Phe Thr Gln Thr Thr Val Glu Gln Ser Gly
290 295 300
Ala Gly Arg Asp Leu Glu Ser Tyr Arg Gln Leu Leu Gly Gln Ala Phe
305 310 315 320
Ala Arg Gly Asn Arg Pro Val Arg Leu Ile Gly Val Gly Val Arg Leu
325 330 335
Leu Asp Leu Gln Gly Ala His Glu Gln Leu Arg Leu Phe
340 345
<210> 6
<211> 358
<212> PRT
<213> Pigmentiphaga sp. H8
<400> 6
Met Arg Lys Ile Ile His Cys Asp Cys Asp Cys Phe Tyr Ala Ser Ile
1 5 10 15
Glu Met Arg Asp Asp Pro Ser Leu Arg Gly Arg Pro Leu Ala Val Gly
20 25 30
Gly Arg Pro Glu Thr Arg Gly Val Val Ala Thr Cys Asn Tyr Glu Ala
35 40 45
Arg Lys Tyr Gly Val His Ser Ala Met Ser Ser Ala Arg Ala Val Arg
50 55 60
Leu Cys Pro Asp Leu Leu Ile Ile Pro Pro Arg Met Glu Met Tyr Arg
65 70 75 80
Val Ala Ser Ala Gln Ile Met Asp Ile Tyr Arg Asp Tyr Thr Glu Leu
85 90 95
Val Glu Pro Leu Ser Leu Asp Glu Ala Tyr Leu Asp Val Thr Gly Ser
100 105 110
Asp Arg Leu Gln Gly Ser Ala Thr Arg Ile Ala Ser Glu Ile Arg Gln
115 120 125
Arg Val Ala Gln Ala Val Gly Ile Thr Val Ser Ala Gly Val Ala Pro
130 135 140
Ser Lys Phe Val Ala Lys Ile Ala Ser Asp Trp Asn Lys Pro Asp Gly
145 150 155 160
Leu Phe Val Val Arg Pro Gln Asp Val Asp Thr Phe Val Ala Ala Leu
165 170 175
Pro Val Ala Lys Leu His Gly Val Gly Lys Val Thr Gly Ala Arg Leu
180 185 190
Lys Ala Leu Gly Val Glu Thr Cys Ala Asp Leu Arg Glu Trp Glu His
195 200 205
Asp Arg Leu Arg Asp Glu Phe Gly Ala Phe Gly Glu Arg Leu His Asp
210 215 220
Leu Cys Arg Gly Ile Asp Leu Arg Glu Val Ser Pro Thr Arg Glu Arg
225 230 235 240
Lys Ser Val Ser Val Glu Gln Thr Phe Val Thr Asp Leu His Thr Leu
245 250 255
Glu Ala Cys Gln Ala Leu Leu Arg Glu Met Leu Asp Gln Leu Asp Ala
260 265 270
Arg Val Arg Arg Ala Asp Ala Gln Asn His Ile Gln Lys Leu Phe Val
275 280 285
Lys Leu Arg Phe Ser Asp Phe Asn Arg Thr Thr Ala Glu Gly Val Gly
290 295 300
Ala Ala Leu Asp Glu Glu Gln Phe Arg Ile Leu Leu Ala Thr Ala Phe
305 310 315 320
Arg Arg Asn Pro Arg Ala Val Arg Leu Met Gly Leu Gly Val Arg Leu
325 330 335
Gly Ala Pro Gly Gly Gln Leu Ala Leu Phe Gly Asp Gln Pro Thr Val
340 345 350
Ser Glu Pro Asp Thr Val
355
<210> 7
<211> 502
<212> PRT
<213> Xenopus tropicalis
<400> 7
Met Ser Phe Ile Pro Leu Lys Arg Arg Arg Ala Gly Pro Val Ser Glu
1 5 10 15
Glu Pro Leu Asp Ser Leu Gln Ser Leu Phe Pro Asp Val Cys Leu Phe
20 25 30
Leu Val Glu Arg Arg Met Gly Ser Ala Arg Arg Lys Phe Leu Thr Gly
35 40 45
Leu Ala Gln Lys Lys Gly Phe Cys Val Thr Pro Gln Phe Ser Asp Gln
50 55 60
Val Thr His Val Val Ser Glu Gln Asn Ser Cys Ser Glu Val Leu Leu
65 70 75 80
Trp Ile Glu Arg Gln Ser Gly Gln Lys Val Gln Pro Gly Gly Ala Glu
85 90 95
Met Thr Pro His Ile Leu Asp Ile Thr Trp Phe Thr Glu Ser Met Ser
100 105 110
Leu Gly Lys Pro Val Lys Val Glu Pro Arg His Cys Leu Gly Val Ser
115 120 125
Asp Ser Ser Val Ser Arg Asp Lys Ala Thr Gln Glu Ile Pro Ala Tyr
130 135 140
Gly Cys Gln Arg Arg Thr Pro Leu His His His Asn Lys Glu Ile Thr
145 150 155 160
Asp Ala Leu Glu Ile Leu Ala Leu Ser Ala Ser Phe Gln Gly Ser Glu
165 170 175
Ala Arg Phe Leu Gly Phe Thr Arg Ala Ser Ser Val Leu Lys Ser Leu
180 185 190
Pro Phe Arg Leu Gln Ser Val Glu Glu Val Lys Asp Leu Pro Trp Cys
195 200 205
Gly Gly His Ser Gln Thr Val Ile Gln Glu Ile Leu Glu Asp Gly Val
210 215 220
Cys Arg Glu Val Glu Thr Val Lys Asn Ser Glu His Phe Gln Ser Met
225 230 235 240
Lys Ala Leu Thr Ser Ile Phe Gly Val Gly Ile Arg Thr Ala Asp Lys
245 250 255
Trp Tyr Arg Asp Gly Val Arg Ser Leu Ser Asp Leu Asn Asn Leu Gly
260 265 270
Gly Lys Leu Thr Ala Glu Gln Lys Ala Gly Leu Leu His Tyr Thr Asp
275 280 285
Leu Gln Gln Ser Val Thr Arg Glu Glu Ala Gly Thr Val Glu Gln Leu
290 295 300
Ile Lys Gly Ala Leu Gln Ser Phe Val Pro Asp Val Arg Val Thr Met
305 310 315 320
Thr Gly Gly Phe Arg Arg Gly Lys Gln Glu Gly His Asp Val Asp Phe
325 330 335
Leu Ile Thr His Pro Asp Glu Glu Ala Leu Asn Gly Leu Leu Arg Lys
340 345 350
Ala Val Ala Trp Leu Asp Gly Lys Gly Ser Val Leu Tyr Tyr His Val
355 360 365
Arg Ala Arg Ser Gln Asn Phe Ser Gly Ser Asn Thr Met Asp Gly His
370 375 380
Glu Thr Cys Tyr Ser Ile Ile Ala Leu Pro Asn Val Cys Pro Glu Lys
385 390 395 400
Pro Ser Pro Asp Ala Glu Lys Ile Glu Pro Asp Leu Asp Lys Asn Ser
405 410 415
Leu Arg Asn Trp Lys Ala Val Arg Val Asp Leu Val Val Cys Pro Tyr
420 425 430
Ser Glu Tyr Phe Tyr Ala Leu Leu Gly Trp Thr Gly Ser Lys His Phe
435 440 445
Glu Arg Glu Leu Arg Arg Phe Ser Leu His Val Lys Lys Met Ser Leu
450 455 460
Asn Ser His Gly Leu Phe Asp Ile Gln Lys Lys Cys His His Pro Ala
465 470 475 480
Thr Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Pro Tyr Val Pro
485 490 495
Pro Ser Glu Arg Asn Ala
500
<210> 8
<211> 530
<212> PRT
<213> Wickerhamomyces ciferrii
<400> 8
Met Asn Arg Ser Gly Gln Val Leu Ser Lys Met Ser Lys Thr Tyr Leu
1 5 10 15
Phe Asp Gly Leu Glu Phe Leu Phe Ile Pro Asn Ile Asn Ser Ser Lys
20 25 30
Val Thr Phe Thr Arg Lys Asn Leu Ala Arg Asn Gly Gly Ala Ser Val
35 40 45
Ala Lys Lys Phe Asp Gln Asp Thr Thr Thr His Val Leu Val Asp Thr
50 55 60
Lys Val Tyr Leu Thr Lys Asp Lys Ile Ser Ala Gly Leu Lys Asn Ala
65 70 75 80
Lys Val Pro Lys Thr Phe Gln Pro Gly Lys Ile Leu Asn Gln Thr Trp
85 90 95
Leu Val Asp Ser Ile Glu Gln Gln Lys Leu Leu Asp Thr Lys Glu Tyr
100 105 110
Ile Ile Lys Leu Asp Glu Leu Lys Pro Glu Thr Arg Lys Glu Ser Pro
115 120 125
Ala Ser Lys Gln His Ile Glu Asn Leu Gln Lys Gln Glu Thr Lys Glu
130 135 140
Lys Leu Ile Ala Glu Ser Ser Thr Gly Asn Pro Asn Glu Arg Thr Ile
145 150 155 160
Phe Leu Leu Asn Gln Met Ala Glu Glu Arg Leu Leu Gln Gly Glu His
165 170 175
Phe Lys Ala Lys Ala Tyr Lys Asn Ala Ile Asn Ala Leu Asn Asn Thr
180 185 190
Gly Asp Phe Ile Ser Asp Ala Asn Glu Ala Leu Arg Leu Lys Gly Ile
195 200 205
Gly Val Ser Val Ala Gln Lys Ile Glu Glu Ile Val Lys Thr Asn Thr
210 215 220
Leu Ser Ser Leu Asn Glu Ile Lys Ser Asp Lys Glu His Gln Val Ser
225 230 235 240
Lys Leu Phe Met Gly Ile His Gly Val Gly Pro Val Ser Ala Lys Lys
245 250 255
Trp Tyr Asn Asp Gly Leu Arg Thr Leu Glu Asp Val Ser Gln Lys Pro
260 265 270
Asp Leu Thr Ser Asn Gln Thr Leu Gly Leu Lys Tyr Tyr Asp Glu Trp
275 280 285
Leu Glu Arg Ile Pro Arg Asp Glu Cys Thr Leu His Asn Glu Phe Met
290 295 300
Ser Asp Leu Val Ser Gln Ile Asp Pro Leu Val Gln Phe Thr Ile Gly
305 310 315 320
Gly Ser Tyr Arg Arg Gly Ser Pro Thr Cys Gly Asp Val Asp Phe Ile
325 330 335
Ile Thr Lys Pro Asn Ala Asp Asn Glu Glu Met Lys Glu Ile Leu Glu
340 345 350
Lys Ile Leu Val Lys Ile Glu Gln Val Gly Tyr Leu Lys Cys Ser Leu
355 360 365
Gln Lys Lys His Ser Thr Lys Phe Leu Ser Gly Cys Ala Leu Pro Pro
370 375 380
Asn Tyr Ala Ser Arg Leu Pro Glu Tyr Ser Glu Gly Lys Trp Gly Lys
385 390 395 400
Cys Arg Arg Ile Asp Phe Leu Met Val Pro Trp Lys Glu Arg Gly Ala
405 410 415
Ala Phe Ile Tyr Phe Thr Gly Asn Asp Tyr Phe Asn Arg Leu Ile Arg
420 425 430
Leu Lys Ala Val Lys Asn Gly Leu Val Leu Asn Glu Ser Gly Leu Phe
435 440 445
Lys Arg Ile Lys Tyr Val Gln Gly Lys Asn Val Glu Asp Lys Thr Met
450 455 460
Leu Ile Glu Ser Phe Ser Glu Lys Lys Ile Phe Lys Leu Leu Gly Phe
465 470 475 480
Lys Tyr Val Pro Pro Glu Gln Arg Asn Phe Gly Ala Asn Asn Pro Pro
485 490 495
Ser Lys Leu Gly Lys His Leu Asp Gln Phe Arg Ile Asp His Lys Tyr
500 505 510
Phe Asp Lys Val Val Lys Glu Glu Ile Ile Asp Asp Asp Val Ile Glu
515 520 525
Val Asp
530
<210> 9
<211> 520
<212> PRT
<213> Bos taurus
<400> 9
Met Ala Gln Gln Arg Gln His Gln Arg Leu Pro Met Asp Pro Leu Cys
1 5 10 15
Thr Ala Ser Ser Gly Pro Arg Lys Lys Arg Pro Arg Gln Val Gly Ala
20 25 30
Ser Met Ala Ser Pro Pro His Asp Ile Lys Phe Gln Asn Leu Val Leu
35 40 45
Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg Arg Asn Phe Leu Met
50 55 60
Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu Asn Glu Leu Ser Asp
65 70 75 80
Ser Val Thr His Ile Val Ala Glu Asn Asn Ser Gly Ser Glu Val Leu
85 90 95
Glu Trp Leu Gln Val Gln Asn Ile Arg Ala Ser Ser Gln Leu Glu Leu
100 105 110
Leu Asp Val Ser Trp Leu Ile Glu Ser Met Gly Ala Gly Lys Pro Val
115 120 125
Glu Ile Thr Gly Lys His Gln Leu Val Val Arg Thr Asp Tyr Ser Ala
130 135 140
Thr Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro Leu Ala Val Lys Lys
145 150 155 160
Ile Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn
165 170 175
His Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu Asn Ser Glu Phe
180 185 190
Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val
195 200 205
Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr Glu Gly
210 215 220
Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Ile Ile Glu Glu Ile Ile
225 230 235 240
Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg
245 250 255
Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Leu Lys
260 265 270
Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser Leu Ser Lys Ile
275 280 285
Met Ser Asp Lys Thr Leu Lys Phe Thr Lys Met Gln Lys Ala Gly Phe
290 295 300
Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu
305 310 315 320
Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp
325 330 335
Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly
340 345 350
His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu
355 360 365
Glu Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly Leu
370 375 380
Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Leu
385 390 395 400
Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu
405 410 415
Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln
420 425 430
Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys
435 440 445
Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg
450 455 460
Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met
465 470 475 480
Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu
485 490 495
Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr
500 505 510
Ile Glu Pro Trp Glu Arg Asn Ala
515 520
<210> 10
<211> 510
<212> PRT
<213> Mus musculus
<400> 10
Met Asp Pro Leu Gln Ala Val His Leu Gly Pro Arg Lys Lys Arg Pro
1 5 10 15
Arg Gln Leu Gly Thr Pro Val Ala Ser Thr Pro Tyr Asp Ile Arg Phe
20 25 30
Arg Asp Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg
35 40 45
Arg Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu
50 55 60
Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser
65 70 75 80
Gly Ser Asp Val Leu Glu Trp Leu Gln Leu Gln Asn Ile Lys Ala Ser
85 90 95
Ser Glu Leu Glu Leu Leu Asp Ile Ser Trp Leu Ile Glu Cys Met Gly
100 105 110
Ala Gly Lys Pro Val Glu Met Met Gly Arg His Gln Leu Val Val Asn
115 120 125
Arg Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala
130 135 140
Pro Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr
145 150 155 160
Leu Asn Asn Tyr Asn Gln Leu Phe Thr Asp Ala Leu Asp Ile Leu Ala
165 170 175
Glu Asn Asp Glu Leu Arg Glu Asn Glu Gly Ser Cys Leu Ala Phe Met
180 185 190
Arg Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met
195 200 205
Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile
210 215 220
Ile Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Ala Lys Ala Val
225 230 235 240
Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe
245 250 255
Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg
260 265 270
Thr Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu Arg Phe Thr Gln Met
275 280 285
Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn
290 295 300
Arg Pro Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val
305 310 315 320
Thr Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg
325 330 335
Gly Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu
340 345 350
Ala Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe
355 360 365
Trp Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr
370 375 380
Phe Glu Lys Phe Lys Gln Pro Ser Arg Lys Val Asp Ala Leu Asp His
385 390 395 400
Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp His Gly Arg Val His
405 410 415
Ser Glu Lys Ser Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg
420 425 430
Val Asp Leu Val Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu
435 440 445
Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala
450 455 460
Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Arg
465 470 475 480
Thr Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala
485 490 495
His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala
500 505 510
<210> 11
<211> 1923
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS017 sequence with His6 tag
<400> 11
atgcatcatc atcaccatca cggcagcagc aagtttacct ggaaagaact gattcagctg 60
ggtagcccga gcaaagcata tgaaagcagc ctggcatgta ttgcccatat tgatatgaat 120
gcatttttcg cacaggttga gcagatgcgt tgtggtctga gcaaagaaga tccggttgtt 180
tgcgttcagt ggaatagcat tattgcagtt agctatgcag cccgtaaata tggtattagc 240
cgtatggata ccattcaaga ggcactgaaa aaatgcagca atctgattcc gattcatacc 300
gcagttttca aaaaaggcga agatttttgg cagtatcatg atggttgtgg tagctgggtt 360
caagatccgg caaaacaaat ttcagtcgaa gatcataaag ttagcctgga accgtatcgt 420
cgtgaaagcc gtaaagccct gaaaatcttt aaaagcgcat gtgatctggt tgaacgtgca 480
agcattgatg aagtttttct ggatctgggt cgcatttgtt ttaacatgct gatgttcgat 540
aacgagtatg aactgaccgg tgatctgaaa ctgaaagatg cactgagcaa tattcgcgaa 600
gcatttattg gtggcaacta tgatattaac agccatctgc cgctgattcc ggaaaaaatc 660
aaaagcctga aattcgaagg cgacgtgttt aatccggaag gtcgtgatct gattacagat 720
tgggatgatg ttattctggc actgggtagt caggtttgta aaggtattcg tgatagcatc 780
aaagatatcc tgggttatac cacctcatgt ggtctgtcaa gcaccaaaaa tgtttgtaaa 840
ctggccagca actacaaaaa accggatgca cagaccattg tgaaaaatga ttgtctgctg 900
gatttcctgg attgcggcaa atttgaaatt accagctttt ggaccttagg tggtgttctg 960
ggtaaagaat taattgatgt gctggatctg ccgcatgaaa acagcattaa acatattcgt 1020
gaaacctggc ctgataatgc aggtcagctg aaagaatttc tggatgccaa agttaaacag 1080
agcgattatg atcgtagcac cagcaatatt gatccgctga aaaccgcaga tctggccgaa 1140
aaactgttta aactgagccg tggtcgttat ggcctgccgc tgtcaagccg tccggttgtg 1200
aaaagcatga tgagcaataa aaacctgcgt ggcaaaagct gcaatagcat tgttgattgt 1260
attagctggc tggaagtttt ttgtgcagaa ctgaccagcc gtattcagga tctggaacaa 1320
gaatataaca agatcgttat tccgcgtacc gttagcatta gcctgaaaac caaaagctat 1380
gaggtgtatc gtaaaagcgg tccggtggca tataaaggta tcaattttca gagccacgaa 1440
ctgctgaaag tgggtatcaa atttgtgacc gatctggata tcaaaggcaa gaacaaaagt 1500
tattacccgc tgaccaaact gagcatgacc attaccaatt tcgatatcat cgatctgcag 1560
aaaaccgtgg ttgatatgtt tggtaatcag gtgcatacgt ttaaaagcag cgcaggtaaa 1620
gaagatgaag aaaaaaccac cagtagcaaa gccgatgaaa aaaccccgaa actggaatgt 1680
tgtaaatatc aggttacctt caccgatcag aaagcactgc aagaacatgc agattatcat 1740
ctggccctga aactgtctga aggtctgaat ggtgcagaag aaagcagcaa aaatctgagc 1800
tttggtgaaa aacgtctgct gtttagccgt aaacgtccga atagccagca taccgcaaca 1860
ccgcagaaaa aacaggttac cagcagtaaa aacatcctga gcttttttac ccgcaaaaaa 1920
tga 1923
<210> 12
<211> 1521
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS024 sequence with His6 tag
<400> 12
atgcatcatc atcaccatca cggcagcttt catgcaaccg cactgcctcg tatgcgtaaa 60
cgtccgcgtc cggaagaagt tgcctgtccg ggtcgtgaag atgttaaatt tcgtgatgtt 120
cgtctgtacc tggtggaaat gaaaatgggt cgtagccgtc gtagctttct gacccagctg 180
gcacgtagca aaggttttat ggttgaagag gttctgagca atcgtgttac ccatgttgtt 240
agcgaaagca gccaggcacc ggttctgtgg gcatggctga aagaacgtgc accgcaggat 300
ctgccgaata tgcatgttgt gaatattacc tggtttaccg atagcatgcg tgaaagccgt 360
ccggttgcag ttgaaacccg tcatctgatt caggataccc tgcctgcaat tccggaaggt 420
ggtgcaccgg cagccgaagt tagccagtat gcatgtcagc gtcgtaccac caccgataac 480
tataatgttg tttttaccga tgcctttgaa gttctggccg aatgctatga atttaatcag 540
atggatggtc gttgtctggc atttcgtcgt gcagcaagcg ttctgaaaag cctgcctcgt 600
ggtctgagca gcctggaaga aacccatagc ctgccgtgtt taggtggtca tgcaaaagca 660
attattggcg aaattctgca gcatggtcgt gcatttgatg ttgaaaaagt tctgagtgat 720
gaacgctatc agaccctgaa actgtttacc agcgtttatg gtgttggtcc gaaaaccgca 780
gaaaaatggt atcgtagcgg tctgcgtagc ctggatcata ttctggcgga tcagagcatc 840
cagctgaatc atatgcagca gaatggtttt ctgcattatg gtgatattag ccgtgcagtt 900
agcaaagccg aagcacgtgc actgaccaaa gcaattggtg aaaccgttca ggcaattaca 960
ccggatgcac tgctggcact gaccggtggt tttcgtcgcg gtaaagaatt tggtcatgat 1020
gtggatatta tctttaccac gctggaatta ggcatggaag aaaatctgct gctggcagtg 1080
attaaaagtc tggaaaaaca gggtattctg ctgtattgtg attatcaggc aagcaccttt 1140
gatctgacca aactgccgac acatagcttt gaagcaatgg atcattttgc caagtgcttt 1200
ctgattctgc gtctggaagc aagccaggtt gaagaaggcc tgaatagtcc ggttgaagat 1260
attcgtggtt ggcgtgcagt tcgtgttgat ctggttagcc ctccggttga tcgttatgca 1320
tttgcactgt taggttggac cggtagccgt cagtttgaac gtgatctgcg tcgttttgca 1380
cgtaaagaac gtcgtatgct gctggataat catggcctgt atgataaaac caaagaagaa 1440
tttctggcag ccggtacgga aaaagatatt tttgatcatc tgggccttga gtatatggaa 1500
ccgtggcagc gtaatgcata a 1521
<210> 13
<211> 1731
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS029 sequence with His6 tag
<400> 13
atgcatcatc atcaccatca cggcagcggt attctgagcg gcaaaaaatt cctgattctg 60
ccgaatagcc ataccggtag cgttaatatt ctggcaggta ttgttaaaga acaaggtggt 120
tttctggtta gcagcgcaga tcgtctgagc aatgatgttg ttgttctggt gaatgatagc 180
ttcgtggaca aaaccaacaa aattgttaat cgcggtctgt ttctgaaaga atttgaactg 240
gatgcaagcg ttgtttggac ctatgttctg gaaaatgaac tggtttgtct gcgtgttagc 300
ctggttccga gctgggttga aaatggcacc tttcatttta gcgatagcga acgtattatt 360
ctgctggata gcgaaagcca agaacgcgat accaaaaatg ttcagtttca tagcgcaggt 420
aatgaagagg caggtagtga tgatgaaacc gatgttgaag gtaataaaga aagcaccggt 480
gatattaccg atgttagcga taccgcaaca ccgcagctgc agagcagtcc gctgagcaaa 540
tatatcaaac aagaagagga tatcgacaac caggttctga ttaaagcact gggtcgtctg 600
gtgaaaaaat acgaagttaa aggtgatcag tatcgcagcc gtagctatcg tctggcaaaa 660
caggcagttg aaaaatatcc gcataaaatc accagcggta gccaggcaca gcgtcagctg 720
agcaatattg gtagcagcat tgccaaaaaa atccagctgc tgctggacac cggtacactg 780
cctggtctgg aagatccggc aaccgatgaa tatgaaagca gcctgggtta tttcagcgaa 840
tgttatggta ttggtgttcc gatggccaaa aaatggatta ccctgaatat cagcaccttt 900
tatcgtgcag cacgtctgca tccgaaactg tttattagcg attggccgat tctgtatggc 960
tggacctatt atgaagattg gagcaaacgt attccgcgtg atgaagttac cgcacatttt 1020
gagctggtta aagaagaagt tcgtcgcgtt ggtaatggtt gtagcgttga aatgcagggt 1080
agctatgttc gtggtgcacg tgataccggt gatgttgatc tgatgttcta caaagaaaat 1140
tgcgacgatc tggaagaggt taccattggt atggaaaatg ttgcagcaag cctgtatcag 1200
aaaggctata tcaaatgttt tctgctgctg accgataaac tggaacgcat gtttcgtccg 1260
gatattctga gtcgtctgca gaaatgtggt attgccgaaa tcagcaatga acataccttt 1320
cgtaatagcg accgtggcaa aaaactgttt ttcggtgttg aactgccagg cgattatccg 1380
atttatccgt ttgatgataa agacatcctg cagctgaaac cgcaggataa attcatgagc 1440
aaaagcaaag atgccggtca tttttgtcgt cgtctggatt tcttctgttg caaatggtca 1500
gaactgggtg cagcccgtat tcattatacc ggtaataccg attataaccg ttggctgcgt 1560
gttcgtgcaa tggatatggg ttataaactg acccagcatg gcatcttcaa agatgatgta 1620
ctgctggaaa gctttgatga gcgcaaaatc tttgaatatc tgcatgtgcc gtatctgaat 1680
ccggttgatc gtaataaaac cgattgggtg aatatcccga ttccgaaata a 1731
<210> 14
<211> 1617
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS030 sequence with His6 tag
<400> 14
atgcatcatc atcaccatca cggcagcaat cgtagcggtc aggttctgag caaaatgagt 60
aaaacctacc tgtttgatgg cctggaattt ctgtttattc cgaacattaa tagcagcaag 120
gtgaccttta cacgcaaaaa tctggcacgt aatggtggtg caagcgttgc caaaaaattc 180
gatcaggata ccaccacaca tgttctggtt gataccaaag tttatctgac caaagacaaa 240
attagcgcag gtctgaaaaa tgccaaagtg ccgaaaacct ttcagcctgg taaaattctg 300
aatcagacct ggctggttga ttctattgaa cagcagaaac tgctggacac caaagagtat 360
attatcaaac tggatgagct gaaaccggaa acgcgtaaag aaagtccggc aagcaaacag 420
catattgaaa atctgcagaa acaagaaacc aaagagaaac tgattgcaga aagcagcacc 480
ggtaatccga atgaacgtac catttttctg ctgaaccaga tggcagaaga acgtctgctg 540
cagggtgaac attttaaagc aaaagcctat aagaacgcca ttaacgccct gaataatacc 600
ggtgatttta tctcagatgc aaatgaagca ctgcgcctga aaggtattgg tgttagcgtg 660
gcacagaaaa ttgaagaaat tgtgaaaacc aatacgctga gcagcctgaa tgaaatcaaa 720
agcgataaag aacaccaggt gagcaaactg tttatgggta ttcatggtgt tggtccggtt 780
agcgcaaaaa agtggtataa tgatggtctg cgtaccctgg aagatgttag ccagaaaccg 840
gatctgacca gcaatcagac cctgggcctg aaatattacg atgaatggct ggaacgtatt 900
ccgcgtgatg aatgtaccct gcataatgaa tttatgagcg atctggtgag ccagattgat 960
ccgctggttc agtttaccat tggtggtagc tatcgtcgtg gtagcccgac ctgtggtgat 1020
gtggatttta tcattaccaa accgaatgcc gataacgaag agatgaaaga gattctggaa 1080
aagatcctgg tgaaaatcga acaggttggt tatctgaaat gtagcctgca gaaaaaacac 1140
agcaccaaat ttctgagcgg ttgtgcactg cctccgaatt atgcaagccg tctgccggaa 1200
tacagcgaag gtaaatgggg taaatgtcgt cgtattgatt ttctgatggt tccgtggaaa 1260
gaacgtggtg cagcatttat ctattttacc ggcaacgatt atttcaaccg tctgattcgt 1320
ctgaaagccg ttaaaaatgg tctggtgctg aatgaatcag gtctgtttaa acgcatcaaa 1380
tacgtgcagg gtaaaaacgt ggaagataaa accatgctga tcgaaagctt tagcgagaaa 1440
aaaatcttta agctgctggg cttcaaatat gttccgcctg aacagcgtaa ttttggtgca 1500
aataatccgc ctagcaaact gggtaaacat ctggatcagt ttcgcatcga tcacaaatat 1560
ttcgacaaag tggtgaaaga agagatcatt gacgacgatg ttatcgaggt ggattaa 1617
<210> 15
<211> 1074
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS053 sequence with His6 tag
<400> 15
atgcatcatc atcaccatca cggcagccgc aaaatcatcc atattgattg cgattgcttt 60
tacgcagcac tggaaatgcg tgatgatccg agcctgcgtg gtaaagcact ggcagttggt 120
ggtagtccgg ataaacgtgg tgttgttgca acctgtagct atgaagcacg tgcatatggt 180
gttcgtagcg caatggcaat gcgtaccgca ctgaaactgt gtccggatct gctggttgtt 240
cgtccgcgtt ttgatgttta tcgtgcagtt agcaaacaaa tccatgccat ctttcgtgat 300
tataccgatc tgattgaacc gctgagcctg gatgaagcat atctggatgt tagcgcaagt 360
ccgcattttg caggtagcgc aacccgtatt gcacaggata ttcgtcgtcg tgttgcagaa 420
gaactgcgta ttaccgttag tgccggtgtt gcaccgaaca aatttctggc aaaaattgca 480
agcgattggc gtaaaccgga tggtctgttt gttattacac cggaacaggt tgatggtttt 540
gttgccgaac tgccggttgc aaaactgcat ggtgttggta aagttaccgc agaacgtctg 600
gcacgtatgg gtattcgtac ctgtgccgat ctgcgtcagg gtagcaaact gagtctggtt 660
cgtgaatttg gtagctttgg tgaacgtctg tggggtttag cacatggtat tgatgaacgt 720
ccggttgaag ttgatagccg tcgtcagagc gttagcgttg aatgtacctt tgatcgtgat 780
ctgccggatc tggcagcatg tctggaagaa ttaccgacac tgctggaaga actggatggt 840
cgtctgcagc gtctggatgg tagctatcgt cctgataaac cgtttgtgaa actgaaattc 900
cacgatttta cccagaccac cgttgaacag agcggtgcag gtcgcgatct ggaaagttat 960
cgtcagctgc tgggtcaagc atttgcacgt ggtaatcgtc cggttcgtct gattggtgtg 1020
ggtgttcgtc tgctggatct gcagggtgca catgaacagc tgcgtctgtt ttaa 1074
<210> 16
<211> 1101
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS054 sequence with His6 tag
<400> 16
atgcatcatc atcaccatca cggcagccgc aaaatcattc attgtgattg cgattgcttt 60
tacgccagca ttgaaatgcg tgatgatccg agcctgcgtg gtcgtccgct ggcagttggt 120
ggccgtccgg aaacacgtgg tgttgttgca acctgtaatt atgaagcacg taaatatggt 180
gttcatagcg caatgagcag cgcacgtgca gttcgtctgt gtccggatct gctgattatt 240
ccgcctcgta tggaaatgta tcgtgttgca agcgcacaga tcatggatat ttatcgtgat 300
tataccgaac tggttgaacc gctgagcctg gatgaagcat atctggatgt taccggtagc 360
gatcgtctgc agggtagcgc aacccgtatt gcaagcgaaa ttcgtcagcg tgttgcacag 420
gccgttggta ttaccgttag tgccggtgtt gcaccgagca aatttgttgc caaaattgcc 480
agcgattgga ataaaccgga tggtctgttt gttgttcgtc cgcaggatgt tgataccttt 540
gttgcagcac tgccggttgc aaaactgcat ggtgttggta aagttaccgg tgcacgtctg 600
aaagcactgg gtgttgaaac ctgtgccgat ctgcgtgaat gggaacatga tcgtttacgt 660
gatgaatttg gtgcatttgg tgaacgtctg cacgatctgt gtcgtggtat tgatctgcgc 720
gaagttagcc cgacacgtga acgtaaaagc gttagcgttg aacagacctt tgttaccgat 780
ctgcataccc tggaagcatg tcaggcactg ctgcgtgaaa tgctggatca gctggatgca 840
cgtgttcgtc gtgcagatgc acagaaccat attcagaaac tgtttgtgaa actgcgcttc 900
agcgatttta atcgtaccac agccgaaggt gttggtgccg cactggatga ggaacagttt 960
cgtattctgc tggcaaccgc atttcgtcgt aatccgcgtg ccgtgcgtct gatgggtctg 1020
ggtgttcgtc tgggtgcacc tggtggtcag ctggcactgt ttggtgatca gccgaccgtt 1080
agcgaaccgg ataccgttta a 1101
<210> 17
<211> 1533
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS066 sequence with His6 tag
<400> 17
atgcatcatc atcaccatca cggcagcagc tttattccgc tgaaacgtcg tcgtgcaggt 60
ccggttagcg aagaaccgct ggatagcctg cagagcctgt ttccggatgt ttgtctgttt 120
ctggttgaac gtcgtatggg tagcgcacgt cgtaaatttc tgaccggtct ggcacagaaa 180
aaaggttttt gtgttacacc gcagtttagc gatcaggtta cccatgttgt tagcgaacag 240
aatagctgta gcgaagttct gctgtggatt gaacgtcaga gtggtcagaa agttcagcct 300
ggtggtgcag aaatgacacc gcatattctg gatattacct ggtttaccga aagcatgagc 360
ctgggtaaac cggttaaagt tgaaccgcgt cattgtctgg gtgttagcga tagcagcgtt 420
agccgtgata aagcaaccca agaaattccg gcatatggtt gtcagcgtcg tacaccgctg 480
catcatcata ataaagaaat taccgatgcg ctggaaattc tggcactgag cgcaagcttt 540
cagggtagcg aagcacgttt tctgggtttt acccgtgcaa gcagcgttct gaaaagcctg 600
ccgtttcgtc tgcagagcgt tgaagaggtt aaagatctgc cgtggtgtgg tggtcatagc 660
cagaccgtta ttcaagaaat cctggaagat ggtgtttgcc gtgaagttga aaccgtgaaa 720
aatagcgaac atttccagag catgaaagca ctgaccagca tttttggtgt tggtattcgt 780
accgcagata aatggtatcg tgatggtgtt cgtagcctga gcgatctgaa taatcttggt 840
ggtaaactga ccgcagaaca gaaagcaggt ctgctgcatt acaccgatct gcagcagagc 900
gtgacccgtg aagaagcagg caccgttgaa cagctgatta aaggtgcact gcagagcttt 960
gtgccggatg tgcgtgttac catgaccggt ggttttcgtc gtggtaaaca agagggtcat 1020
gatgtggatt ttctgattac ccatcctgat gaagaagccc tgaacggcct gctgcgtaaa 1080
gcagttgcat ggctggatgg taaaggtagc gttctgtatt atcatgttcg tgcacgtagt 1140
cagaatttta gcggtagcaa taccatggat ggtcatgaaa cctgttatag cattattgca 1200
ctgccgaatg tttgtccgga aaaaccgagt ccggatgcag aaaaaattga accggatctg 1260
gataaaaaca gcctgcgtaa ttggaaagca gttcgtgttg atctggttgt ttgcccgtat 1320
agcgaatact tttatgcact gttaggttgg accggcagca aacattttga acgtgaactg 1380
cgtcgtttta gcctgcatgt gaaaaaaatg agcctgaata gccatggcct gtttgacatt 1440
cagaaaaagt gtcatcatcc ggcaaccagc gaagaagaaa tttttgcaca tctgggtctg 1500
ccgtatgttc cgcctagcga acgtaatgca taa 1533
<210> 18
<211> 1317
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS082 sequence with His6 tag
<400> 18
atgcatcatc atcaccatca cggcagcgaa cagcagaaac tgctggacac caaagagtat 60
attatcaaac tggatgagct gaaaccggaa acgcgtaaag aaagtccggc aagcaaacag 120
catattgaaa atctgcagaa acaagaaacc aaagagaaac tgattgcaga aagcagcacc 180
ggtaatccga atgaacgtac catttttctg ctgaaccaga tggcagaaga acgtctgctg 240
cagggtgaac attttaaagc aaaagcctat aagaacgcca ttaacgccct gaataatacc 300
ggtgatttta tctcagatgc aaatgaagca ctgcgcctga aaggtattgg tgttagcgtg 360
gcacagaaaa ttgaagaaat tgtgaaaacc aatacgctga gcagcctgaa tgaaatcaaa 420
agcgataaag aacaccaggt gagcaaactg tttatgggta ttcatggtgt tggtccggtt 480
agcgcaaaaa agtggtataa tgatggtctg cgtaccctgg aagatgttag ccagaaaccg 540
gatctgacca gcaatcagac cctgggcctg aaatattacg atgaatggct ggaacgtatt 600
ccgcgtgatg aatgtaccct gcataatgaa tttatgagcg atctggtgag ccagattgat 660
ccgctggttc agtttaccat tggtggtagc tatcgtcgtg gtagcccgac ctgtggtgat 720
gtggatttta tcattaccaa accgaatgcc gataacgaag agatgaaaga gattctggaa 780
aagatcctgg tgaaaatcga acaggttggt tatctgaaat gtagcctgca gaaaaaacac 840
agcaccaaat ttctgagcgg ttgtgcactg cctccgaatt atgcaagccg tctgccggaa 900
tacagcgaag gtaaatgggg taaatgtcgt cgtattgatt ttctgatggt tccgtggaaa 960
gaacgtggtg cagcatttat ctattttacc ggcaacgatt atttcaaccg tctgattcgt 1020
ctgaaagccg ttaaaaatgg tctggtgctg aatgaatcag gtctgtttaa acgcatcaaa 1080
tacgtgcagg gtaaaaacgt ggaagataaa accatgctga tcgaaagctt tagcgagaaa 1140
aaaatcttta agctgctggg cttcaaatat gttccgcctg aacagcgtaa ttttggtgca 1200
aataatccgc ctagcaaact gggtaaacat ctggatcagt ttcgcatcga tcacaaatat 1260
ttcgacaaag tggtgaaaga agagatcatt gacgacgatg ttatcgaggt ggattaa 1317
<210> 19
<211> 1176
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS048 sequence with His6 tag
<400> 19
atgcatcatc atcaccatca cggcagccgt accgattata gcgcaacccc gaatccgggt 60
tttcagaaaa caccgcctct ggcagtgaaa aaaatcagcc agtatgcatg tcagcgtaaa 120
accacactga ataactataa ccacatcttc accgatgcct ttgaaattct ggcagaaaac 180
agcgaattca aagaaaacga agttagctac gtgaccttta tgcgtgcagc aagcgttctg 240
aaaagcctgc cgtttaccat tattagcatg aaagataccg aaggtattcc gtgtctgggt 300
gataaagtga aatgcatcat tgaagagatc atcgaagatg gtgaaagcag cgaagttaaa 360
gcagttctga atgatgaacg ttaccagagc ttcaaactgt ttaccagcgt ttttggtgtt 420
ggcctgaaaa ccagcgaaaa atggtttcgt atgggttttc gtagcctgag caaaatcatg 480
agcgataaaa ccctgaaatt caccaaaatg cagaaagccg gtttcctgta ttatgaagat 540
ctggtgagct gtgttacccg tgccgaagcc gaagcagttg gtgttctggt taaagaagca 600
gtttgggcat ttctgccgga tgcatttgtt accatgaccg gtggttttcg tcgtggcaaa 660
aaaatcggtc atgatgtgga ttttctgatt accagtccgg gtagcgcaga agatgaagaa 720
cagctgctgc cgaaagttat taatctgtgg gaaaaaaaag gcctgctgct gtattacgat 780
ctggttgaaa gcaccttcga gaaattcaaa ctgccgagcc gtcaggttga taccctggat 840
cactttcaga aatgttttct tatcctgaag ctgcatcatc agcgtgttga tagcagcaaa 900
agcaatcagc aagaaggtaa aacctggaaa gcaattcgtg ttgatctggt tatgtgcccg 960
tatgaaaatc gtgcatttgc actgttaggt tggaccggta gtcgtcagtt tgaacgtgat 1020
attcgtcgtt atgcaaccca tgaacgtaaa atgatgctgg ataatcatgc cctgtacgat 1080
aaaacgaaac gcgtgttcct gaaagccgaa agcgaagaag aaatttttgc acatctgggc 1140
cttgattaca ttgaaccgtg ggaacgtaat gcctaa 1176
<210> 20
<211> 1554
<212> DNA
<213> Artificial Sequence
<220>
<223> Cloned EDS015 sequence with His6 tag
<400> 20
atgcatcatc atcaccatca cggcagcgat ccgctgcagg cagttcatct gggtccgcgt 60
aaaaaacgtc cgcgtcagct gggtacaccg gttgcaagca ccccgtatga tattcgtttt 120
cgtgatctgg ttctgttcat cctggaaaaa aagatgggta caacccgtcg tgcatttctg 180
atggaactgg cacgtcgtaa aggttttcgt gttgaaaatg aactgagcga tagcgttacc 240
catattgttg cagaaaataa cagcggtagt gatgttctgg aatggctgca actgcagaac 300
attaaagcaa gcagcgaact ggaactgctg gatattagct ggctgattga atgtatgggt 360
gcaggtaaac cggttgaaat gatgggtcgt catcagctgg ttgttaatcg taatagcagc 420
ccgagtccgg ttccgggtag ccagaatgtt ccggcaccgg cagtgaaaaa aatcagtcag 480
tatgcatgtc agcgtcgtac cacactgaat aactataatc agctgtttac cgatgcactg 540
gatattctgg cagaaaatga tgagctgcgc gaaaatgaag gtagctgtct ggcatttatg 600
cgtgccagca gcgttctgaa aagcctgccg tttccgatta ccagcatgaa agataccgaa 660
ggtattccgt gtctgggtga taaagtgaaa agcattattg aaggcatcat cgaagatggc 720
gaaagcagtg aagcaaaagc agttctgaat gatgaacgct acaaaagctt caaactgttt 780
accagcgttt ttggtgttgg tctgaaaacc gcagaaaaat ggtttcgtat gggttttcgt 840
accctgagca aaattcagag cgataaaagt ctgcgtttta cccagatgca gaaagcaggt 900
tttctgtatt atgaagatct ggtgagctgc gttaatcgtc cggaagccga agcagttagc 960
atgctggtta aagaagcagt tgttaccttt ctgccggatg cgctggttac catgaccggt 1020
ggttttcgtc gcggaaaaat gacaggtcat gatgtggatt ttctgattac ctcaccggaa 1080
gcaaccgaag atgaagaaca gcaactgctg cataaagtta ccgatttttg gaaacagcag 1140
ggtctgctgc tgtattgtga tatcctggaa tcaaccttcg agaaattcaa acagccgagc 1200
cgtaaagttg atgccctgga tcattttcag aagtgttttc tgatcctgaa actggatcat 1260
ggtcgtgttc atagcgaaaa aagcggtcag caagaaggta aaggttggaa agcaattcgt 1320
gtggatctgg ttatgtgtcc gtatgatcgt cgtgcctttg cactgttagg ttggaccggt 1380
agccgtcagt ttgaacgtga tctgcgtcgt tatgcaaccc atgaacgtaa aatgatgctg 1440
gataatcatg cactgtatga tcgcaccaaa cgtgtttttc tggaagcaga aagcgaagaa 1500
gaaatctttg cacatctggg ccttgattac attgaaccgt gggaacgtaa tgca 1554
<210> 21
<211> 640
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS017 expressed protein sequence with His6 tag
<400> 21
Met His His His His His His Gly Ser Ser Lys Phe Thr Trp Lys Glu
1 5 10 15
Leu Ile Gln Leu Gly Ser Pro Ser Lys Ala Tyr Glu Ser Ser Leu Ala
20 25 30
Cys Ile Ala His Ile Asp Met Asn Ala Phe Phe Ala Gln Val Glu Gln
35 40 45
Met Arg Cys Gly Leu Ser Lys Glu Asp Pro Val Val Cys Val Gln Trp
50 55 60
Asn Ser Ile Ile Ala Val Ser Tyr Ala Ala Arg Lys Tyr Gly Ile Ser
65 70 75 80
Arg Met Asp Thr Ile Gln Glu Ala Leu Lys Lys Cys Ser Asn Leu Ile
85 90 95
Pro Ile His Thr Ala Val Phe Lys Lys Gly Glu Asp Phe Trp Gln Tyr
100 105 110
His Asp Gly Cys Gly Ser Trp Val Gln Asp Pro Ala Lys Gln Ile Ser
115 120 125
Val Glu Asp His Lys Val Ser Leu Glu Pro Tyr Arg Arg Glu Ser Arg
130 135 140
Lys Ala Leu Lys Ile Phe Lys Ser Ala Cys Asp Leu Val Glu Arg Ala
145 150 155 160
Ser Ile Asp Glu Val Phe Leu Asp Leu Gly Arg Ile Cys Phe Asn Met
165 170 175
Leu Met Phe Asp Asn Glu Tyr Glu Leu Thr Gly Asp Leu Lys Leu Lys
180 185 190
Asp Ala Leu Ser Asn Ile Arg Glu Ala Phe Ile Gly Gly Asn Tyr Asp
195 200 205
Ile Asn Ser His Leu Pro Leu Ile Pro Glu Lys Ile Lys Ser Leu Lys
210 215 220
Phe Glu Gly Asp Val Phe Asn Pro Glu Gly Arg Asp Leu Ile Thr Asp
225 230 235 240
Trp Asp Asp Val Ile Leu Ala Leu Gly Ser Gln Val Cys Lys Gly Ile
245 250 255
Arg Asp Ser Ile Lys Asp Ile Leu Gly Tyr Thr Thr Ser Cys Gly Leu
260 265 270
Ser Ser Thr Lys Asn Val Cys Lys Leu Ala Ser Asn Tyr Lys Lys Pro
275 280 285
Asp Ala Gln Thr Ile Val Lys Asn Asp Cys Leu Leu Asp Phe Leu Asp
290 295 300
Cys Gly Lys Phe Glu Ile Thr Ser Phe Trp Thr Leu Gly Gly Val Leu
305 310 315 320
Gly Lys Glu Leu Ile Asp Val Leu Asp Leu Pro His Glu Asn Ser Ile
325 330 335
Lys His Ile Arg Glu Thr Trp Pro Asp Asn Ala Gly Gln Leu Lys Glu
340 345 350
Phe Leu Asp Ala Lys Val Lys Gln Ser Asp Tyr Asp Arg Ser Thr Ser
355 360 365
Asn Ile Asp Pro Leu Lys Thr Ala Asp Leu Ala Glu Lys Leu Phe Lys
370 375 380
Leu Ser Arg Gly Arg Tyr Gly Leu Pro Leu Ser Ser Arg Pro Val Val
385 390 395 400
Lys Ser Met Met Ser Asn Lys Asn Leu Arg Gly Lys Ser Cys Asn Ser
405 410 415
Ile Val Asp Cys Ile Ser Trp Leu Glu Val Phe Cys Ala Glu Leu Thr
420 425 430
Ser Arg Ile Gln Asp Leu Glu Gln Glu Tyr Asn Lys Ile Val Ile Pro
435 440 445
Arg Thr Val Ser Ile Ser Leu Lys Thr Lys Ser Tyr Glu Val Tyr Arg
450 455 460
Lys Ser Gly Pro Val Ala Tyr Lys Gly Ile Asn Phe Gln Ser His Glu
465 470 475 480
Leu Leu Lys Val Gly Ile Lys Phe Val Thr Asp Leu Asp Ile Lys Gly
485 490 495
Lys Asn Lys Ser Tyr Tyr Pro Leu Thr Lys Leu Ser Met Thr Ile Thr
500 505 510
Asn Phe Asp Ile Ile Asp Leu Gln Lys Thr Val Val Asp Met Phe Gly
515 520 525
Asn Gln Val His Thr Phe Lys Ser Ser Ala Gly Lys Glu Asp Glu Glu
530 535 540
Lys Thr Thr Ser Ser Lys Ala Asp Glu Lys Thr Pro Lys Leu Glu Cys
545 550 555 560
Cys Lys Tyr Gln Val Thr Phe Thr Asp Gln Lys Ala Leu Gln Glu His
565 570 575
Ala Asp Tyr His Leu Ala Leu Lys Leu Ser Glu Gly Leu Asn Gly Ala
580 585 590
Glu Glu Ser Ser Lys Asn Leu Ser Phe Gly Glu Lys Arg Leu Leu Phe
595 600 605
Ser Arg Lys Arg Pro Asn Ser Gln His Thr Ala Thr Pro Gln Lys Lys
610 615 620
Gln Val Thr Ser Ser Lys Asn Ile Leu Ser Phe Phe Thr Arg Lys Lys
625 630 635 640
<210> 22
<211> 506
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS024 expressed protein sequence with His6 tag
<400> 22
Met His His His His His His Gly Ser Phe His Ala Thr Ala Leu Pro
1 5 10 15
Arg Met Arg Lys Arg Pro Arg Pro Glu Glu Val Ala Cys Pro Gly Arg
20 25 30
Glu Asp Val Lys Phe Arg Asp Val Arg Leu Tyr Leu Val Glu Met Lys
35 40 45
Met Gly Arg Ser Arg Arg Ser Phe Leu Thr Gln Leu Ala Arg Ser Lys
50 55 60
Gly Phe Met Val Glu Glu Val Leu Ser Asn Arg Val Thr His Val Val
65 70 75 80
Ser Glu Ser Ser Gln Ala Pro Val Leu Trp Ala Trp Leu Lys Glu Arg
85 90 95
Ala Pro Gln Asp Leu Pro Asn Met His Val Val Asn Ile Thr Trp Phe
100 105 110
Thr Asp Ser Met Arg Glu Ser Arg Pro Val Ala Val Glu Thr Arg His
115 120 125
Leu Ile Gln Asp Thr Leu Pro Ala Ile Pro Glu Gly Gly Ala Pro Ala
130 135 140
Ala Glu Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Thr Asp Asn
145 150 155 160
Tyr Asn Val Val Phe Thr Asp Ala Phe Glu Val Leu Ala Glu Cys Tyr
165 170 175
Glu Phe Asn Gln Met Asp Gly Arg Cys Leu Ala Phe Arg Arg Ala Ala
180 185 190
Ser Val Leu Lys Ser Leu Pro Arg Gly Leu Ser Ser Leu Glu Glu Thr
195 200 205
His Ser Leu Pro Cys Leu Gly Gly His Ala Lys Ala Ile Ile Gly Glu
210 215 220
Ile Leu Gln His Gly Arg Ala Phe Asp Val Glu Lys Val Leu Ser Asp
225 230 235 240
Glu Arg Tyr Gln Thr Leu Lys Leu Phe Thr Ser Val Tyr Gly Val Gly
245 250 255
Pro Lys Thr Ala Glu Lys Trp Tyr Arg Ser Gly Leu Arg Ser Leu Asp
260 265 270
His Ile Leu Ala Asp Gln Ser Ile Gln Leu Asn His Met Gln Gln Asn
275 280 285
Gly Phe Leu His Tyr Gly Asp Ile Ser Arg Ala Val Ser Lys Ala Glu
290 295 300
Ala Arg Ala Leu Thr Lys Ala Ile Gly Glu Thr Val Gln Ala Ile Thr
305 310 315 320
Pro Asp Ala Leu Leu Ala Leu Thr Gly Gly Phe Arg Arg Gly Lys Glu
325 330 335
Phe Gly His Asp Val Asp Ile Ile Phe Thr Thr Leu Glu Leu Gly Met
340 345 350
Glu Glu Asn Leu Leu Leu Ala Val Ile Lys Ser Leu Glu Lys Gln Gly
355 360 365
Ile Leu Leu Tyr Cys Asp Tyr Gln Ala Ser Thr Phe Asp Leu Thr Lys
370 375 380
Leu Pro Thr His Ser Phe Glu Ala Met Asp His Phe Ala Lys Cys Phe
385 390 395 400
Leu Ile Leu Arg Leu Glu Ala Ser Gln Val Glu Glu Gly Leu Asn Ser
405 410 415
Pro Val Glu Asp Ile Arg Gly Trp Arg Ala Val Arg Val Asp Leu Val
420 425 430
Ser Pro Pro Val Asp Arg Tyr Ala Phe Ala Leu Leu Gly Trp Thr Gly
435 440 445
Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Phe Ala Arg Lys Glu Arg
450 455 460
Arg Met Leu Leu Asp Asn His Gly Leu Tyr Asp Lys Thr Lys Glu Glu
465 470 475 480
Phe Leu Ala Ala Gly Thr Glu Lys Asp Ile Phe Asp His Leu Gly Leu
485 490 495
Glu Tyr Met Glu Pro Trp Gln Arg Asn Ala
500 505
<210> 23
<211> 576
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS029 expressed protein sequence with His6 tag
<400> 23
Met His His His His His His Gly Ser Gly Ile Leu Ser Gly Lys Lys
1 5 10 15
Phe Leu Ile Leu Pro Asn Ser His Thr Gly Ser Val Asn Ile Leu Ala
20 25 30
Gly Ile Val Lys Glu Gln Gly Gly Phe Leu Val Ser Ser Ala Asp Arg
35 40 45
Leu Ser Asn Asp Val Val Val Leu Val Asn Asp Ser Phe Val Asp Lys
50 55 60
Thr Asn Lys Ile Val Asn Arg Gly Leu Phe Leu Lys Glu Phe Glu Leu
65 70 75 80
Asp Ala Ser Val Val Trp Thr Tyr Val Leu Glu Asn Glu Leu Val Cys
85 90 95
Leu Arg Val Ser Leu Val Pro Ser Trp Val Glu Asn Gly Thr Phe His
100 105 110
Phe Ser Asp Ser Glu Arg Ile Ile Leu Leu Asp Ser Glu Ser Gln Glu
115 120 125
Arg Asp Thr Lys Asn Val Gln Phe His Ser Ala Gly Asn Glu Glu Ala
130 135 140
Gly Ser Asp Asp Glu Thr Asp Val Glu Gly Asn Lys Glu Ser Thr Gly
145 150 155 160
Asp Ile Thr Asp Val Ser Asp Thr Ala Thr Pro Gln Leu Gln Ser Ser
165 170 175
Pro Leu Ser Lys Tyr Ile Lys Gln Glu Glu Asp Ile Asp Asn Gln Val
180 185 190
Leu Ile Lys Ala Leu Gly Arg Leu Val Lys Lys Tyr Glu Val Lys Gly
195 200 205
Asp Gln Tyr Arg Ser Arg Ser Tyr Arg Leu Ala Lys Gln Ala Val Glu
210 215 220
Lys Tyr Pro His Lys Ile Thr Ser Gly Ser Gln Ala Gln Arg Gln Leu
225 230 235 240
Ser Asn Ile Gly Ser Ser Ile Ala Lys Lys Ile Gln Leu Leu Leu Asp
245 250 255
Thr Gly Thr Leu Pro Gly Leu Glu Asp Pro Ala Thr Asp Glu Tyr Glu
260 265 270
Ser Ser Leu Gly Tyr Phe Ser Glu Cys Tyr Gly Ile Gly Val Pro Met
275 280 285
Ala Lys Lys Trp Ile Thr Leu Asn Ile Ser Thr Phe Tyr Arg Ala Ala
290 295 300
Arg Leu His Pro Lys Leu Phe Ile Ser Asp Trp Pro Ile Leu Tyr Gly
305 310 315 320
Trp Thr Tyr Tyr Glu Asp Trp Ser Lys Arg Ile Pro Arg Asp Glu Val
325 330 335
Thr Ala His Phe Glu Leu Val Lys Glu Glu Val Arg Arg Val Gly Asn
340 345 350
Gly Cys Ser Val Glu Met Gln Gly Ser Tyr Val Arg Gly Ala Arg Asp
355 360 365
Thr Gly Asp Val Asp Leu Met Phe Tyr Lys Glu Asn Cys Asp Asp Leu
370 375 380
Glu Glu Val Thr Ile Gly Met Glu Asn Val Ala Ala Ser Leu Tyr Gln
385 390 395 400
Lys Gly Tyr Ile Lys Cys Phe Leu Leu Leu Thr Asp Lys Leu Glu Arg
405 410 415
Met Phe Arg Pro Asp Ile Leu Ser Arg Leu Gln Lys Cys Gly Ile Ala
420 425 430
Glu Ile Ser Asn Glu His Thr Phe Arg Asn Ser Asp Arg Gly Lys Lys
435 440 445
Leu Phe Phe Gly Val Glu Leu Pro Gly Asp Tyr Pro Ile Tyr Pro Phe
450 455 460
Asp Asp Lys Asp Ile Leu Gln Leu Lys Pro Gln Asp Lys Phe Met Ser
465 470 475 480
Lys Ser Lys Asp Ala Gly His Phe Cys Arg Arg Leu Asp Phe Phe Cys
485 490 495
Cys Lys Trp Ser Glu Leu Gly Ala Ala Arg Ile His Tyr Thr Gly Asn
500 505 510
Thr Asp Tyr Asn Arg Trp Leu Arg Val Arg Ala Met Asp Met Gly Tyr
515 520 525
Lys Leu Thr Gln His Gly Ile Phe Lys Asp Asp Val Leu Leu Glu Ser
530 535 540
Phe Asp Glu Arg Lys Ile Phe Glu Tyr Leu His Val Pro Tyr Leu Asn
545 550 555 560
Pro Val Asp Arg Asn Lys Thr Asp Trp Val Asn Ile Pro Ile Pro Lys
565 570 575
<210> 24
<211> 538
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS030 expressed protein sequence with His6 tag
<400> 24
Met His His His His His His Gly Ser Asn Arg Ser Gly Gln Val Leu
1 5 10 15
Ser Lys Met Ser Lys Thr Tyr Leu Phe Asp Gly Leu Glu Phe Leu Phe
20 25 30
Ile Pro Asn Ile Asn Ser Ser Lys Val Thr Phe Thr Arg Lys Asn Leu
35 40 45
Ala Arg Asn Gly Gly Ala Ser Val Ala Lys Lys Phe Asp Gln Asp Thr
50 55 60
Thr Thr His Val Leu Val Asp Thr Lys Val Tyr Leu Thr Lys Asp Lys
65 70 75 80
Ile Ser Ala Gly Leu Lys Asn Ala Lys Val Pro Lys Thr Phe Gln Pro
85 90 95
Gly Lys Ile Leu Asn Gln Thr Trp Leu Val Asp Ser Ile Glu Gln Gln
100 105 110
Lys Leu Leu Asp Thr Lys Glu Tyr Ile Ile Lys Leu Asp Glu Leu Lys
115 120 125
Pro Glu Thr Arg Lys Glu Ser Pro Ala Ser Lys Gln His Ile Glu Asn
130 135 140
Leu Gln Lys Gln Glu Thr Lys Glu Lys Leu Ile Ala Glu Ser Ser Thr
145 150 155 160
Gly Asn Pro Asn Glu Arg Thr Ile Phe Leu Leu Asn Gln Met Ala Glu
165 170 175
Glu Arg Leu Leu Gln Gly Glu His Phe Lys Ala Lys Ala Tyr Lys Asn
180 185 190
Ala Ile Asn Ala Leu Asn Asn Thr Gly Asp Phe Ile Ser Asp Ala Asn
195 200 205
Glu Ala Leu Arg Leu Lys Gly Ile Gly Val Ser Val Ala Gln Lys Ile
210 215 220
Glu Glu Ile Val Lys Thr Asn Thr Leu Ser Ser Leu Asn Glu Ile Lys
225 230 235 240
Ser Asp Lys Glu His Gln Val Ser Lys Leu Phe Met Gly Ile His Gly
245 250 255
Val Gly Pro Val Ser Ala Lys Lys Trp Tyr Asn Asp Gly Leu Arg Thr
260 265 270
Leu Glu Asp Val Ser Gln Lys Pro Asp Leu Thr Ser Asn Gln Thr Leu
275 280 285
Gly Leu Lys Tyr Tyr Asp Glu Trp Leu Glu Arg Ile Pro Arg Asp Glu
290 295 300
Cys Thr Leu His Asn Glu Phe Met Ser Asp Leu Val Ser Gln Ile Asp
305 310 315 320
Pro Leu Val Gln Phe Thr Ile Gly Gly Ser Tyr Arg Arg Gly Ser Pro
325 330 335
Thr Cys Gly Asp Val Asp Phe Ile Ile Thr Lys Pro Asn Ala Asp Asn
340 345 350
Glu Glu Met Lys Glu Ile Leu Glu Lys Ile Leu Val Lys Ile Glu Gln
355 360 365
Val Gly Tyr Leu Lys Cys Ser Leu Gln Lys Lys His Ser Thr Lys Phe
370 375 380
Leu Ser Gly Cys Ala Leu Pro Pro Asn Tyr Ala Ser Arg Leu Pro Glu
385 390 395 400
Tyr Ser Glu Gly Lys Trp Gly Lys Cys Arg Arg Ile Asp Phe Leu Met
405 410 415
Val Pro Trp Lys Glu Arg Gly Ala Ala Phe Ile Tyr Phe Thr Gly Asn
420 425 430
Asp Tyr Phe Asn Arg Leu Ile Arg Leu Lys Ala Val Lys Asn Gly Leu
435 440 445
Val Leu Asn Glu Ser Gly Leu Phe Lys Arg Ile Lys Tyr Val Gln Gly
450 455 460
Lys Asn Val Glu Asp Lys Thr Met Leu Ile Glu Ser Phe Ser Glu Lys
465 470 475 480
Lys Ile Phe Lys Leu Leu Gly Phe Lys Tyr Val Pro Pro Glu Gln Arg
485 490 495
Asn Phe Gly Ala Asn Asn Pro Pro Ser Lys Leu Gly Lys His Leu Asp
500 505 510
Gln Phe Arg Ile Asp His Lys Tyr Phe Asp Lys Val Val Lys Glu Glu
515 520 525
Ile Ile Asp Asp Asp Val Ile Glu Val Asp
530 535
<210> 25
<211> 357
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS053 expressed protein sequence with His6 tag
<400> 25
Met His His His His His His Gly Ser Arg Lys Ile Ile His Ile Asp
1 5 10 15
Cys Asp Cys Phe Tyr Ala Ala Leu Glu Met Arg Asp Asp Pro Ser Leu
20 25 30
Arg Gly Lys Ala Leu Ala Val Gly Gly Ser Pro Asp Lys Arg Gly Val
35 40 45
Val Ala Thr Cys Ser Tyr Glu Ala Arg Ala Tyr Gly Val Arg Ser Ala
50 55 60
Met Ala Met Arg Thr Ala Leu Lys Leu Cys Pro Asp Leu Leu Val Val
65 70 75 80
Arg Pro Arg Phe Asp Val Tyr Arg Ala Val Ser Lys Gln Ile His Ala
85 90 95
Ile Phe Arg Asp Tyr Thr Asp Leu Ile Glu Pro Leu Ser Leu Asp Glu
100 105 110
Ala Tyr Leu Asp Val Ser Ala Ser Pro His Phe Ala Gly Ser Ala Thr
115 120 125
Arg Ile Ala Gln Asp Ile Arg Arg Arg Val Ala Glu Glu Leu Arg Ile
130 135 140
Thr Val Ser Ala Gly Val Ala Pro Asn Lys Phe Leu Ala Lys Ile Ala
145 150 155 160
Ser Asp Trp Arg Lys Pro Asp Gly Leu Phe Val Ile Thr Pro Glu Gln
165 170 175
Val Asp Gly Phe Val Ala Glu Leu Pro Val Ala Lys Leu His Gly Val
180 185 190
Gly Lys Val Thr Ala Glu Arg Leu Ala Arg Met Gly Ile Arg Thr Cys
195 200 205
Ala Asp Leu Arg Gln Gly Ser Lys Leu Ser Leu Val Arg Glu Phe Gly
210 215 220
Ser Phe Gly Glu Arg Leu Trp Gly Leu Ala His Gly Ile Asp Glu Arg
225 230 235 240
Pro Val Glu Val Asp Ser Arg Arg Gln Ser Val Ser Val Glu Cys Thr
245 250 255
Phe Asp Arg Asp Leu Pro Asp Leu Ala Ala Cys Leu Glu Glu Leu Pro
260 265 270
Thr Leu Leu Glu Glu Leu Asp Gly Arg Leu Gln Arg Leu Asp Gly Ser
275 280 285
Tyr Arg Pro Asp Lys Pro Phe Val Lys Leu Lys Phe His Asp Phe Thr
290 295 300
Gln Thr Thr Val Glu Gln Ser Gly Ala Gly Arg Asp Leu Glu Ser Tyr
305 310 315 320
Arg Gln Leu Leu Gly Gln Ala Phe Ala Arg Gly Asn Arg Pro Val Arg
325 330 335
Leu Ile Gly Val Gly Val Arg Leu Leu Asp Leu Gln Gly Ala His Glu
340 345 350
Gln Leu Arg Leu Phe
355
<210> 26
<211> 366
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS054 expressed protein sequence with His6 tag
<400> 26
Met His His His His His His Gly Ser Arg Lys Ile Ile His Cys Asp
1 5 10 15
Cys Asp Cys Phe Tyr Ala Ser Ile Glu Met Arg Asp Asp Pro Ser Leu
20 25 30
Arg Gly Arg Pro Leu Ala Val Gly Gly Arg Pro Glu Thr Arg Gly Val
35 40 45
Val Ala Thr Cys Asn Tyr Glu Ala Arg Lys Tyr Gly Val His Ser Ala
50 55 60
Met Ser Ser Ala Arg Ala Val Arg Leu Cys Pro Asp Leu Leu Ile Ile
65 70 75 80
Pro Pro Arg Met Glu Met Tyr Arg Val Ala Ser Ala Gln Ile Met Asp
85 90 95
Ile Tyr Arg Asp Tyr Thr Glu Leu Val Glu Pro Leu Ser Leu Asp Glu
100 105 110
Ala Tyr Leu Asp Val Thr Gly Ser Asp Arg Leu Gln Gly Ser Ala Thr
115 120 125
Arg Ile Ala Ser Glu Ile Arg Gln Arg Val Ala Gln Ala Val Gly Ile
130 135 140
Thr Val Ser Ala Gly Val Ala Pro Ser Lys Phe Val Ala Lys Ile Ala
145 150 155 160
Ser Asp Trp Asn Lys Pro Asp Gly Leu Phe Val Val Arg Pro Gln Asp
165 170 175
Val Asp Thr Phe Val Ala Ala Leu Pro Val Ala Lys Leu His Gly Val
180 185 190
Gly Lys Val Thr Gly Ala Arg Leu Lys Ala Leu Gly Val Glu Thr Cys
195 200 205
Ala Asp Leu Arg Glu Trp Glu His Asp Arg Leu Arg Asp Glu Phe Gly
210 215 220
Ala Phe Gly Glu Arg Leu His Asp Leu Cys Arg Gly Ile Asp Leu Arg
225 230 235 240
Glu Val Ser Pro Thr Arg Glu Arg Lys Ser Val Ser Val Glu Gln Thr
245 250 255
Phe Val Thr Asp Leu His Thr Leu Glu Ala Cys Gln Ala Leu Leu Arg
260 265 270
Glu Met Leu Asp Gln Leu Asp Ala Arg Val Arg Arg Ala Asp Ala Gln
275 280 285
Asn His Ile Gln Lys Leu Phe Val Lys Leu Arg Phe Ser Asp Phe Asn
290 295 300
Arg Thr Thr Ala Glu Gly Val Gly Ala Ala Leu Asp Glu Glu Gln Phe
305 310 315 320
Arg Ile Leu Leu Ala Thr Ala Phe Arg Arg Asn Pro Arg Ala Val Arg
325 330 335
Leu Met Gly Leu Gly Val Arg Leu Gly Ala Pro Gly Gly Gln Leu Ala
340 345 350
Leu Phe Gly Asp Gln Pro Thr Val Ser Glu Pro Asp Thr Val
355 360 365
<210> 27
<211> 510
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS066 expressed protein sequence with His6 tag
<400> 27
Met His His His His His His Gly Ser Ser Phe Ile Pro Leu Lys Arg
1 5 10 15
Arg Arg Ala Gly Pro Val Ser Glu Glu Pro Leu Asp Ser Leu Gln Ser
20 25 30
Leu Phe Pro Asp Val Cys Leu Phe Leu Val Glu Arg Arg Met Gly Ser
35 40 45
Ala Arg Arg Lys Phe Leu Thr Gly Leu Ala Gln Lys Lys Gly Phe Cys
50 55 60
Val Thr Pro Gln Phe Ser Asp Gln Val Thr His Val Val Ser Glu Gln
65 70 75 80
Asn Ser Cys Ser Glu Val Leu Leu Trp Ile Glu Arg Gln Ser Gly Gln
85 90 95
Lys Val Gln Pro Gly Gly Ala Glu Met Thr Pro His Ile Leu Asp Ile
100 105 110
Thr Trp Phe Thr Glu Ser Met Ser Leu Gly Lys Pro Val Lys Val Glu
115 120 125
Pro Arg His Cys Leu Gly Val Ser Asp Ser Ser Val Ser Arg Asp Lys
130 135 140
Ala Thr Gln Glu Ile Pro Ala Tyr Gly Cys Gln Arg Arg Thr Pro Leu
145 150 155 160
His His His Asn Lys Glu Ile Thr Asp Ala Leu Glu Ile Leu Ala Leu
165 170 175
Ser Ala Ser Phe Gln Gly Ser Glu Ala Arg Phe Leu Gly Phe Thr Arg
180 185 190
Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Arg Leu Gln Ser Val Glu
195 200 205
Glu Val Lys Asp Leu Pro Trp Cys Gly Gly His Ser Gln Thr Val Ile
210 215 220
Gln Glu Ile Leu Glu Asp Gly Val Cys Arg Glu Val Glu Thr Val Lys
225 230 235 240
Asn Ser Glu His Phe Gln Ser Met Lys Ala Leu Thr Ser Ile Phe Gly
245 250 255
Val Gly Ile Arg Thr Ala Asp Lys Trp Tyr Arg Asp Gly Val Arg Ser
260 265 270
Leu Ser Asp Leu Asn Asn Leu Gly Gly Lys Leu Thr Ala Glu Gln Lys
275 280 285
Ala Gly Leu Leu His Tyr Thr Asp Leu Gln Gln Ser Val Thr Arg Glu
290 295 300
Glu Ala Gly Thr Val Glu Gln Leu Ile Lys Gly Ala Leu Gln Ser Phe
305 310 315 320
Val Pro Asp Val Arg Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys
325 330 335
Gln Glu Gly His Asp Val Asp Phe Leu Ile Thr His Pro Asp Glu Glu
340 345 350
Ala Leu Asn Gly Leu Leu Arg Lys Ala Val Ala Trp Leu Asp Gly Lys
355 360 365
Gly Ser Val Leu Tyr Tyr His Val Arg Ala Arg Ser Gln Asn Phe Ser
370 375 380
Gly Ser Asn Thr Met Asp Gly His Glu Thr Cys Tyr Ser Ile Ile Ala
385 390 395 400
Leu Pro Asn Val Cys Pro Glu Lys Pro Ser Pro Asp Ala Glu Lys Ile
405 410 415
Glu Pro Asp Leu Asp Lys Asn Ser Leu Arg Asn Trp Lys Ala Val Arg
420 425 430
Val Asp Leu Val Val Cys Pro Tyr Ser Glu Tyr Phe Tyr Ala Leu Leu
435 440 445
Gly Trp Thr Gly Ser Lys His Phe Glu Arg Glu Leu Arg Arg Phe Ser
450 455 460
Leu His Val Lys Lys Met Ser Leu Asn Ser His Gly Leu Phe Asp Ile
465 470 475 480
Gln Lys Lys Cys His His Pro Ala Thr Ser Glu Glu Glu Ile Phe Ala
485 490 495
His Leu Gly Leu Pro Tyr Val Pro Pro Ser Glu Arg Asn Ala
500 505 510
<210> 28
<211> 438
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS082 expressed protein sequence with His6 tag
<400> 28
Met His His His His His His Gly Ser Glu Gln Gln Lys Leu Leu Asp
1 5 10 15
Thr Lys Glu Tyr Ile Ile Lys Leu Asp Glu Leu Lys Pro Glu Thr Arg
20 25 30
Lys Glu Ser Pro Ala Ser Lys Gln His Ile Glu Asn Leu Gln Lys Gln
35 40 45
Glu Thr Lys Glu Lys Leu Ile Ala Glu Ser Ser Thr Gly Asn Pro Asn
50 55 60
Glu Arg Thr Ile Phe Leu Leu Asn Gln Met Ala Glu Glu Arg Leu Leu
65 70 75 80
Gln Gly Glu His Phe Lys Ala Lys Ala Tyr Lys Asn Ala Ile Asn Ala
85 90 95
Leu Asn Asn Thr Gly Asp Phe Ile Ser Asp Ala Asn Glu Ala Leu Arg
100 105 110
Leu Lys Gly Ile Gly Val Ser Val Ala Gln Lys Ile Glu Glu Ile Val
115 120 125
Lys Thr Asn Thr Leu Ser Ser Leu Asn Glu Ile Lys Ser Asp Lys Glu
130 135 140
His Gln Val Ser Lys Leu Phe Met Gly Ile His Gly Val Gly Pro Val
145 150 155 160
Ser Ala Lys Lys Trp Tyr Asn Asp Gly Leu Arg Thr Leu Glu Asp Val
165 170 175
Ser Gln Lys Pro Asp Leu Thr Ser Asn Gln Thr Leu Gly Leu Lys Tyr
180 185 190
Tyr Asp Glu Trp Leu Glu Arg Ile Pro Arg Asp Glu Cys Thr Leu His
195 200 205
Asn Glu Phe Met Ser Asp Leu Val Ser Gln Ile Asp Pro Leu Val Gln
210 215 220
Phe Thr Ile Gly Gly Ser Tyr Arg Arg Gly Ser Pro Thr Cys Gly Asp
225 230 235 240
Val Asp Phe Ile Ile Thr Lys Pro Asn Ala Asp Asn Glu Glu Met Lys
245 250 255
Glu Ile Leu Glu Lys Ile Leu Val Lys Ile Glu Gln Val Gly Tyr Leu
260 265 270
Lys Cys Ser Leu Gln Lys Lys His Ser Thr Lys Phe Leu Ser Gly Cys
275 280 285
Ala Leu Pro Pro Asn Tyr Ala Ser Arg Leu Pro Glu Tyr Ser Glu Gly
290 295 300
Lys Trp Gly Lys Cys Arg Arg Ile Asp Phe Leu Met Val Pro Trp Lys
305 310 315 320
Glu Arg Gly Ala Ala Phe Ile Tyr Phe Thr Gly Asn Asp Tyr Phe Asn
325 330 335
Arg Leu Ile Arg Leu Lys Ala Val Lys Asn Gly Leu Val Leu Asn Glu
340 345 350
Ser Gly Leu Phe Lys Arg Ile Lys Tyr Val Gln Gly Lys Asn Val Glu
355 360 365
Asp Lys Thr Met Leu Ile Glu Ser Phe Ser Glu Lys Lys Ile Phe Lys
370 375 380
Leu Leu Gly Phe Lys Tyr Val Pro Pro Glu Gln Arg Asn Phe Gly Ala
385 390 395 400
Asn Asn Pro Pro Ser Lys Leu Gly Lys His Leu Asp Gln Phe Arg Ile
405 410 415
Asp His Lys Tyr Phe Asp Lys Val Val Lys Glu Glu Ile Ile Asp Asp
420 425 430
Asp Val Ile Glu Val Asp
435
<210> 29
<211> 391
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS048 expressed protein sequence with His6 tag
<400> 29
Met His His His His His His Gly Ser Arg Thr Asp Tyr Ser Ala Thr
1 5 10 15
Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro Leu Ala Val Lys Lys Ile
20 25 30
Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His
35 40 45
Ile Phe Thr Asp Ala Phe Glu Ile Leu Ala Glu Asn Ser Glu Phe Lys
50 55 60
Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu
65 70 75 80
Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys Asp Thr Glu Gly Ile
85 90 95
Pro Cys Leu Gly Asp Lys Val Lys Cys Ile Ile Glu Glu Ile Ile Glu
100 105 110
Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr
115 120 125
Gln Ser Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Leu Lys Thr
130 135 140
Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser Leu Ser Lys Ile Met
145 150 155 160
Ser Asp Lys Thr Leu Lys Phe Thr Lys Met Gln Lys Ala Gly Phe Leu
165 170 175
Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala
180 185 190
Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala
195 200 205
Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His
210 215 220
Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu
225 230 235 240
Gln Leu Leu Pro Lys Val Ile Asn Leu Trp Glu Lys Lys Gly Leu Leu
245 250 255
Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Leu Pro
260 265 270
Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile
275 280 285
Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln
290 295 300
Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro
305 310 315 320
Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln
325 330 335
Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met
340 345 350
Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys
355 360 365
Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile
370 375 380
Glu Pro Trp Glu Arg Asn Ala
385 390
<210> 30
<211> 518
<212> PRT
<213> Artificial Sequence
<220>
<223> EDS015 expressed protein sequence with His6 tag
<400> 30
Met His His His His His His Gly Ser Asp Pro Leu Gln Ala Val His
1 5 10 15
Leu Gly Pro Arg Lys Lys Arg Pro Arg Gln Leu Gly Thr Pro Val Ala
20 25 30
Ser Thr Pro Tyr Asp Ile Arg Phe Arg Asp Leu Val Leu Phe Ile Leu
35 40 45
Glu Lys Lys Met Gly Thr Thr Arg Arg Ala Phe Leu Met Glu Leu Ala
50 55 60
Arg Arg Lys Gly Phe Arg Val Glu Asn Glu Leu Ser Asp Ser Val Thr
65 70 75 80
His Ile Val Ala Glu Asn Asn Ser Gly Ser Asp Val Leu Glu Trp Leu
85 90 95
Gln Leu Gln Asn Ile Lys Ala Ser Ser Glu Leu Glu Leu Leu Asp Ile
100 105 110
Ser Trp Leu Ile Glu Cys Met Gly Ala Gly Lys Pro Val Glu Met Met
115 120 125
Gly Arg His Gln Leu Val Val Asn Arg Asn Ser Ser Pro Ser Pro Val
130 135 140
Pro Gly Ser Gln Asn Val Pro Ala Pro Ala Val Lys Lys Ile Ser Gln
145 150 155 160
Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn Asn Tyr Asn Gln Leu Phe
165 170 175
Thr Asp Ala Leu Asp Ile Leu Ala Glu Asn Asp Glu Leu Arg Glu Asn
180 185 190
Glu Gly Ser Cys Leu Ala Phe Met Arg Ala Ser Ser Val Leu Lys Ser
195 200 205
Leu Pro Phe Pro Ile Thr Ser Met Lys Asp Thr Glu Gly Ile Pro Cys
210 215 220
Leu Gly Asp Lys Val Lys Ser Ile Ile Glu Gly Ile Ile Glu Asp Gly
225 230 235 240
Glu Ser Ser Glu Ala Lys Ala Val Leu Asn Asp Glu Arg Tyr Lys Ser
245 250 255
Phe Lys Leu Phe Thr Ser Val Phe Gly Val Gly Leu Lys Thr Ala Glu
260 265 270
Lys Trp Phe Arg Met Gly Phe Arg Thr Leu Ser Lys Ile Gln Ser Asp
275 280 285
Lys Ser Leu Arg Phe Thr Gln Met Gln Lys Ala Gly Phe Leu Tyr Tyr
290 295 300
Glu Asp Leu Val Ser Cys Val Asn Arg Pro Glu Ala Glu Ala Val Ser
305 310 315 320
Met Leu Val Lys Glu Ala Val Val Thr Phe Leu Pro Asp Ala Leu Val
325 330 335
Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Met Thr Gly His Asp Val
340 345 350
Asp Phe Leu Ile Thr Ser Pro Glu Ala Thr Glu Asp Glu Glu Gln Gln
355 360 365
Leu Leu His Lys Val Thr Asp Phe Trp Lys Gln Gln Gly Leu Leu Leu
370 375 380
Tyr Cys Asp Ile Leu Glu Ser Thr Phe Glu Lys Phe Lys Gln Pro Ser
385 390 395 400
Arg Lys Val Asp Ala Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu
405 410 415
Lys Leu Asp His Gly Arg Val His Ser Glu Lys Ser Gly Gln Gln Glu
420 425 430
Gly Lys Gly Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr
435 440 445
Asp Arg Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe
450 455 460
Glu Arg Asp Leu Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu
465 470 475 480
Asp Asn His Ala Leu Tyr Asp Arg Thr Lys Arg Val Phe Leu Glu Ala
485 490 495
Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu
500 505 510
Pro Trp Glu Arg Asn Ala
515
<210> 31
<211> 5515
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1077 expression vector full sequence
<400> 31
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcagc aagtttacct ggaaagaact 1560
gattcagctg ggtagcccga gcaaagcata tgaaagcagc ctggcatgta ttgcccatat 1620
tgatatgaat gcatttttcg cacaggttga gcagatgcgt tgtggtctga gcaaagaaga 1680
tccggttgtt tgcgttcagt ggaatagcat tattgcagtt agctatgcag cccgtaaata 1740
tggtattagc cgtatggata ccattcaaga ggcactgaaa aaatgcagca atctgattcc 1800
gattcatacc gcagttttca aaaaaggcga agatttttgg cagtatcatg atggttgtgg 1860
tagctgggtt caagatccgg caaaacaaat ttcagtcgaa gatcataaag ttagcctgga 1920
accgtatcgt cgtgaaagcc gtaaagccct gaaaatcttt aaaagcgcat gtgatctggt 1980
tgaacgtgca agcattgatg aagtttttct ggatctgggt cgcatttgtt ttaacatgct 2040
gatgttcgat aacgagtatg aactgaccgg tgatctgaaa ctgaaagatg cactgagcaa 2100
tattcgcgaa gcatttattg gtggcaacta tgatattaac agccatctgc cgctgattcc 2160
ggaaaaaatc aaaagcctga aattcgaagg cgacgtgttt aatccggaag gtcgtgatct 2220
gattacagat tgggatgatg ttattctggc actgggtagt caggtttgta aaggtattcg 2280
tgatagcatc aaagatatcc tgggttatac cacctcatgt ggtctgtcaa gcaccaaaaa 2340
tgtttgtaaa ctggccagca actacaaaaa accggatgca cagaccattg tgaaaaatga 2400
ttgtctgctg gatttcctgg attgcggcaa atttgaaatt accagctttt ggaccttagg 2460
tggtgttctg ggtaaagaat taattgatgt gctggatctg ccgcatgaaa acagcattaa 2520
acatattcgt gaaacctggc ctgataatgc aggtcagctg aaagaatttc tggatgccaa 2580
agttaaacag agcgattatg atcgtagcac cagcaatatt gatccgctga aaaccgcaga 2640
tctggccgaa aaactgttta aactgagccg tggtcgttat ggcctgccgc tgtcaagccg 2700
tccggttgtg aaaagcatga tgagcaataa aaacctgcgt ggcaaaagct gcaatagcat 2760
tgttgattgt attagctggc tggaagtttt ttgtgcagaa ctgaccagcc gtattcagga 2820
tctggaacaa gaatataaca agatcgttat tccgcgtacc gttagcatta gcctgaaaac 2880
caaaagctat gaggtgtatc gtaaaagcgg tccggtggca tataaaggta tcaattttca 2940
gagccacgaa ctgctgaaag tgggtatcaa atttgtgacc gatctggata tcaaaggcaa 3000
gaacaaaagt tattacccgc tgaccaaact gagcatgacc attaccaatt tcgatatcat 3060
cgatctgcag aaaaccgtgg ttgatatgtt tggtaatcag gtgcatacgt ttaaaagcag 3120
cgcaggtaaa gaagatgaag aaaaaaccac cagtagcaaa gccgatgaaa aaaccccgaa 3180
actggaatgt tgtaaatatc aggttacctt caccgatcag aaagcactgc aagaacatgc 3240
agattatcat ctggccctga aactgtctga aggtctgaat ggtgcagaag aaagcagcaa 3300
aaatctgagc tttggtgaaa aacgtctgct gtttagccgt aaacgtccga atagccagca 3360
taccgcaaca ccgcagaaaa aacaggttac cagcagtaaa aacatcctga gcttttttac 3420
ccgcaaaaaa tgatgcacgt gaggatccaa ctcgagaact tagatggtat tagtgacctg 3480
taacagagca ttagcgcaag gtgatttttg tcttcttgcg ctaatttttt gtcatcaaac 3540
ctgtcgctag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 3600
ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 3660
tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct 3720
atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 3780
gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 3840
gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 3900
tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3960
tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4020
gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4080
acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4140
tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4200
gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4260
tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 4320
accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 4380
ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 4440
agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 4500
gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 4560
ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 4620
tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 4680
ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 4740
gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 4800
acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4860
aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4920
atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4980
gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5040
tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5100
ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5160
ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5220
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5280
aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5340
cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5400
gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5460
ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaa 5515
<210> 32
<211> 5113
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1084 expression vector full sequence
<400> 32
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcttt catgcaaccg cactgcctcg 1560
tatgcgtaaa cgtccgcgtc cggaagaagt tgcctgtccg ggtcgtgaag atgttaaatt 1620
tcgtgatgtt cgtctgtacc tggtggaaat gaaaatgggt cgtagccgtc gtagctttct 1680
gacccagctg gcacgtagca aaggttttat ggttgaagag gttctgagca atcgtgttac 1740
ccatgttgtt agcgaaagca gccaggcacc ggttctgtgg gcatggctga aagaacgtgc 1800
accgcaggat ctgccgaata tgcatgttgt gaatattacc tggtttaccg atagcatgcg 1860
tgaaagccgt ccggttgcag ttgaaacccg tcatctgatt caggataccc tgcctgcaat 1920
tccggaaggt ggtgcaccgg cagccgaagt tagccagtat gcatgtcagc gtcgtaccac 1980
caccgataac tataatgttg tttttaccga tgcctttgaa gttctggccg aatgctatga 2040
atttaatcag atggatggtc gttgtctggc atttcgtcgt gcagcaagcg ttctgaaaag 2100
cctgcctcgt ggtctgagca gcctggaaga aacccatagc ctgccgtgtt taggtggtca 2160
tgcaaaagca attattggcg aaattctgca gcatggtcgt gcatttgatg ttgaaaaagt 2220
tctgagtgat gaacgctatc agaccctgaa actgtttacc agcgtttatg gtgttggtcc 2280
gaaaaccgca gaaaaatggt atcgtagcgg tctgcgtagc ctggatcata ttctggcgga 2340
tcagagcatc cagctgaatc atatgcagca gaatggtttt ctgcattatg gtgatattag 2400
ccgtgcagtt agcaaagccg aagcacgtgc actgaccaaa gcaattggtg aaaccgttca 2460
ggcaattaca ccggatgcac tgctggcact gaccggtggt tttcgtcgcg gtaaagaatt 2520
tggtcatgat gtggatatta tctttaccac gctggaatta ggcatggaag aaaatctgct 2580
gctggcagtg attaaaagtc tggaaaaaca gggtattctg ctgtattgtg attatcaggc 2640
aagcaccttt gatctgacca aactgccgac acatagcttt gaagcaatgg atcattttgc 2700
caagtgcttt ctgattctgc gtctggaagc aagccaggtt gaagaaggcc tgaatagtcc 2760
ggttgaagat attcgtggtt ggcgtgcagt tcgtgttgat ctggttagcc ctccggttga 2820
tcgttatgca tttgcactgt taggttggac cggtagccgt cagtttgaac gtgatctgcg 2880
tcgttttgca cgtaaagaac gtcgtatgct gctggataat catggcctgt atgataaaac 2940
caaagaagaa tttctggcag ccggtacgga aaaagatatt tttgatcatc tgggccttga 3000
gtatatggaa ccgtggcagc gtaatgcata atgcacgtga ggatccaact cgagaactta 3060
gatggtatta gtgacctgta acagagcatt agcgcaaggt gatttttgtc ttcttgcgct 3120
aattttttgt catcaaacct gtcgctagtt aagccagccc cgacacccgc caacacccgc 3180
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 3240
ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 3300
gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 3360
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 3420
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 3480
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 3540
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 3600
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 3660
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 3720
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 3780
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 3840
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 3900
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 3960
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 4020
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 4080
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 4140
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 4200
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 4260
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 4320
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 4380
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 4440
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 4500
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 4560
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 4620
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 4680
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 4740
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 4800
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 4860
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 4920
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 4980
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 5040
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 5100
gagcctatgg aaa 5113
<210> 33
<211> 5323
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1089 expression vector full sequence
<400> 33
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcggt attctgagcg gcaaaaaatt 1560
cctgattctg ccgaatagcc ataccggtag cgttaatatt ctggcaggta ttgttaaaga 1620
acaaggtggt tttctggtta gcagcgcaga tcgtctgagc aatgatgttg ttgttctggt 1680
gaatgatagc ttcgtggaca aaaccaacaa aattgttaat cgcggtctgt ttctgaaaga 1740
atttgaactg gatgcaagcg ttgtttggac ctatgttctg gaaaatgaac tggtttgtct 1800
gcgtgttagc ctggttccga gctgggttga aaatggcacc tttcatttta gcgatagcga 1860
acgtattatt ctgctggata gcgaaagcca agaacgcgat accaaaaatg ttcagtttca 1920
tagcgcaggt aatgaagagg caggtagtga tgatgaaacc gatgttgaag gtaataaaga 1980
aagcaccggt gatattaccg atgttagcga taccgcaaca ccgcagctgc agagcagtcc 2040
gctgagcaaa tatatcaaac aagaagagga tatcgacaac caggttctga ttaaagcact 2100
gggtcgtctg gtgaaaaaat acgaagttaa aggtgatcag tatcgcagcc gtagctatcg 2160
tctggcaaaa caggcagttg aaaaatatcc gcataaaatc accagcggta gccaggcaca 2220
gcgtcagctg agcaatattg gtagcagcat tgccaaaaaa atccagctgc tgctggacac 2280
cggtacactg cctggtctgg aagatccggc aaccgatgaa tatgaaagca gcctgggtta 2340
tttcagcgaa tgttatggta ttggtgttcc gatggccaaa aaatggatta ccctgaatat 2400
cagcaccttt tatcgtgcag cacgtctgca tccgaaactg tttattagcg attggccgat 2460
tctgtatggc tggacctatt atgaagattg gagcaaacgt attccgcgtg atgaagttac 2520
cgcacatttt gagctggtta aagaagaagt tcgtcgcgtt ggtaatggtt gtagcgttga 2580
aatgcagggt agctatgttc gtggtgcacg tgataccggt gatgttgatc tgatgttcta 2640
caaagaaaat tgcgacgatc tggaagaggt taccattggt atggaaaatg ttgcagcaag 2700
cctgtatcag aaaggctata tcaaatgttt tctgctgctg accgataaac tggaacgcat 2760
gtttcgtccg gatattctga gtcgtctgca gaaatgtggt attgccgaaa tcagcaatga 2820
acataccttt cgtaatagcg accgtggcaa aaaactgttt ttcggtgttg aactgccagg 2880
cgattatccg atttatccgt ttgatgataa agacatcctg cagctgaaac cgcaggataa 2940
attcatgagc aaaagcaaag atgccggtca tttttgtcgt cgtctggatt tcttctgttg 3000
caaatggtca gaactgggtg cagcccgtat tcattatacc ggtaataccg attataaccg 3060
ttggctgcgt gttcgtgcaa tggatatggg ttataaactg acccagcatg gcatcttcaa 3120
agatgatgta ctgctggaaa gctttgatga gcgcaaaatc tttgaatatc tgcatgtgcc 3180
gtatctgaat ccggttgatc gtaataaaac cgattgggtg aatatcccga ttccgaaata 3240
atgcacgtga ggatccaact cgagaactta gatggtatta gtgacctgta acagagcatt 3300
agcgcaaggt gatttttgtc ttcttgcgct aattttttgt catcaaacct gtcgctagtt 3360
aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 3420
ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 3480
accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 3540
taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 3600
cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 3660
ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 3720
ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 3780
aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 3840
actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 3900
gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 3960
agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 4020
cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 4080
catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 4140
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 4200
gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 4260
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 4320
agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 4380
ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 4440
actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 4500
aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 4560
gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 4620
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 4680
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 4740
tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 4800
ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 4860
agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 4920
ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 4980
tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 5040
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 5100
cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 5160
ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 5220
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 5280
tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaa 5323
<210> 34
<211> 5209
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1090 expression vector full sequence
<400> 34
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcaat cgtagcggtc aggttctgag 1560
caaaatgagt aaaacctacc tgtttgatgg cctggaattt ctgtttattc cgaacattaa 1620
tagcagcaag gtgaccttta cacgcaaaaa tctggcacgt aatggtggtg caagcgttgc 1680
caaaaaattc gatcaggata ccaccacaca tgttctggtt gataccaaag tttatctgac 1740
caaagacaaa attagcgcag gtctgaaaaa tgccaaagtg ccgaaaacct ttcagcctgg 1800
taaaattctg aatcagacct ggctggttga ttctattgaa cagcagaaac tgctggacac 1860
caaagagtat attatcaaac tggatgagct gaaaccggaa acgcgtaaag aaagtccggc 1920
aagcaaacag catattgaaa atctgcagaa acaagaaacc aaagagaaac tgattgcaga 1980
aagcagcacc ggtaatccga atgaacgtac catttttctg ctgaaccaga tggcagaaga 2040
acgtctgctg cagggtgaac attttaaagc aaaagcctat aagaacgcca ttaacgccct 2100
gaataatacc ggtgatttta tctcagatgc aaatgaagca ctgcgcctga aaggtattgg 2160
tgttagcgtg gcacagaaaa ttgaagaaat tgtgaaaacc aatacgctga gcagcctgaa 2220
tgaaatcaaa agcgataaag aacaccaggt gagcaaactg tttatgggta ttcatggtgt 2280
tggtccggtt agcgcaaaaa agtggtataa tgatggtctg cgtaccctgg aagatgttag 2340
ccagaaaccg gatctgacca gcaatcagac cctgggcctg aaatattacg atgaatggct 2400
ggaacgtatt ccgcgtgatg aatgtaccct gcataatgaa tttatgagcg atctggtgag 2460
ccagattgat ccgctggttc agtttaccat tggtggtagc tatcgtcgtg gtagcccgac 2520
ctgtggtgat gtggatttta tcattaccaa accgaatgcc gataacgaag agatgaaaga 2580
gattctggaa aagatcctgg tgaaaatcga acaggttggt tatctgaaat gtagcctgca 2640
gaaaaaacac agcaccaaat ttctgagcgg ttgtgcactg cctccgaatt atgcaagccg 2700
tctgccggaa tacagcgaag gtaaatgggg taaatgtcgt cgtattgatt ttctgatggt 2760
tccgtggaaa gaacgtggtg cagcatttat ctattttacc ggcaacgatt atttcaaccg 2820
tctgattcgt ctgaaagccg ttaaaaatgg tctggtgctg aatgaatcag gtctgtttaa 2880
acgcatcaaa tacgtgcagg gtaaaaacgt ggaagataaa accatgctga tcgaaagctt 2940
tagcgagaaa aaaatcttta agctgctggg cttcaaatat gttccgcctg aacagcgtaa 3000
ttttggtgca aataatccgc ctagcaaact gggtaaacat ctggatcagt ttcgcatcga 3060
tcacaaatat ttcgacaaag tggtgaaaga agagatcatt gacgacgatg ttatcgaggt 3120
ggattaatgc acgtgaggat ccaactcgag aacttagatg gtattagtga cctgtaacag 3180
agcattagcg caaggtgatt tttgtcttct tgcgctaatt ttttgtcatc aaacctgtcg 3240
ctagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3300
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3360
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3420
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3480
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3540
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3600
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3660
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3720
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3780
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 3840
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 3900
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 3960
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 4020
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 4080
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 4140
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 4200
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 4260
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4320
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4380
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4440
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4500
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4560
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4620
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4680
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4740
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4800
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4860
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4920
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4980
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 5040
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 5100
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 5160
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 5209
<210> 35
<211> 4666
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1113 expression vector full sequence
<400> 35
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagccgc aaaatcatcc atattgattg 1560
cgattgcttt tacgcagcac tggaaatgcg tgatgatccg agcctgcgtg gtaaagcact 1620
ggcagttggt ggtagtccgg ataaacgtgg tgttgttgca acctgtagct atgaagcacg 1680
tgcatatggt gttcgtagcg caatggcaat gcgtaccgca ctgaaactgt gtccggatct 1740
gctggttgtt cgtccgcgtt ttgatgttta tcgtgcagtt agcaaacaaa tccatgccat 1800
ctttcgtgat tataccgatc tgattgaacc gctgagcctg gatgaagcat atctggatgt 1860
tagcgcaagt ccgcattttg caggtagcgc aacccgtatt gcacaggata ttcgtcgtcg 1920
tgttgcagaa gaactgcgta ttaccgttag tgccggtgtt gcaccgaaca aatttctggc 1980
aaaaattgca agcgattggc gtaaaccgga tggtctgttt gttattacac cggaacaggt 2040
tgatggtttt gttgccgaac tgccggttgc aaaactgcat ggtgttggta aagttaccgc 2100
agaacgtctg gcacgtatgg gtattcgtac ctgtgccgat ctgcgtcagg gtagcaaact 2160
gagtctggtt cgtgaatttg gtagctttgg tgaacgtctg tggggtttag cacatggtat 2220
tgatgaacgt ccggttgaag ttgatagccg tcgtcagagc gttagcgttg aatgtacctt 2280
tgatcgtgat ctgccggatc tggcagcatg tctggaagaa ttaccgacac tgctggaaga 2340
actggatggt cgtctgcagc gtctggatgg tagctatcgt cctgataaac cgtttgtgaa 2400
actgaaattc cacgatttta cccagaccac cgttgaacag agcggtgcag gtcgcgatct 2460
ggaaagttat cgtcagctgc tgggtcaagc atttgcacgt ggtaatcgtc cggttcgtct 2520
gattggtgtg ggtgttcgtc tgctggatct gcagggtgca catgaacagc tgcgtctgtt 2580
ttaatgcacg tgaggatcca actcgagaac ttagatggta ttagtgacct gtaacagagc 2640
attagcgcaa ggtgattttt gtcttcttgc gctaattttt tgtcatcaaa cctgtcgcta 2700
gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 2760
cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 2820
ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 2880
ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 2940
gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 3000
acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 3060
tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 3120
agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 3180
cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 3240
aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 3300
gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 3360
agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 3420
aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 3480
gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 3540
ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 3600
aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 3660
aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 3720
tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 3780
agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 3840
ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 3900
ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 3960
ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 4020
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 4080
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 4140
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4200
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 4260
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 4320
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 4380
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 4440
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 4500
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 4560
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 4620
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaa 4666
<210> 36
<211> 4693
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1114 expression vector full sequence
<400> 36
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagccgc aaaatcattc attgtgattg 1560
cgattgcttt tacgccagca ttgaaatgcg tgatgatccg agcctgcgtg gtcgtccgct 1620
ggcagttggt ggccgtccgg aaacacgtgg tgttgttgca acctgtaatt atgaagcacg 1680
taaatatggt gttcatagcg caatgagcag cgcacgtgca gttcgtctgt gtccggatct 1740
gctgattatt ccgcctcgta tggaaatgta tcgtgttgca agcgcacaga tcatggatat 1800
ttatcgtgat tataccgaac tggttgaacc gctgagcctg gatgaagcat atctggatgt 1860
taccggtagc gatcgtctgc agggtagcgc aacccgtatt gcaagcgaaa ttcgtcagcg 1920
tgttgcacag gccgttggta ttaccgttag tgccggtgtt gcaccgagca aatttgttgc 1980
caaaattgcc agcgattgga ataaaccgga tggtctgttt gttgttcgtc cgcaggatgt 2040
tgataccttt gttgcagcac tgccggttgc aaaactgcat ggtgttggta aagttaccgg 2100
tgcacgtctg aaagcactgg gtgttgaaac ctgtgccgat ctgcgtgaat gggaacatga 2160
tcgtttacgt gatgaatttg gtgcatttgg tgaacgtctg cacgatctgt gtcgtggtat 2220
tgatctgcgc gaagttagcc cgacacgtga acgtaaaagc gttagcgttg aacagacctt 2280
tgttaccgat ctgcataccc tggaagcatg tcaggcactg ctgcgtgaaa tgctggatca 2340
gctggatgca cgtgttcgtc gtgcagatgc acagaaccat attcagaaac tgtttgtgaa 2400
actgcgcttc agcgatttta atcgtaccac agccgaaggt gttggtgccg cactggatga 2460
ggaacagttt cgtattctgc tggcaaccgc atttcgtcgt aatccgcgtg ccgtgcgtct 2520
gatgggtctg ggtgttcgtc tgggtgcacc tggtggtcag ctggcactgt ttggtgatca 2580
gccgaccgtt agcgaaccgg ataccgttta atgcacgtga ggatccaact cgagaactta 2640
gatggtatta gtgacctgta acagagcatt agcgcaaggt gatttttgtc ttcttgcgct 2700
aattttttgt catcaaacct gtcgctagtt aagccagccc cgacacccgc caacacccgc 2760
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 2820
ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 2880
gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 2940
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 3000
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 3060
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 3120
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 3180
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 3240
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 3300
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 3360
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 3420
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 3480
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 3540
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 3600
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 3660
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 3720
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 3780
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 3840
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 3900
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 3960
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 4020
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 4080
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 4140
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 4200
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 4260
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 4320
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 4380
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 4440
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 4500
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 4560
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 4620
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 4680
gagcctatgg aaa 4693
<210> 37
<211> 5125
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1126 expression vector full sequence
<400> 37
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcagc tttattccgc tgaaacgtcg 1560
tcgtgcaggt ccggttagcg aagaaccgct ggatagcctg cagagcctgt ttccggatgt 1620
ttgtctgttt ctggttgaac gtcgtatggg tagcgcacgt cgtaaatttc tgaccggtct 1680
ggcacagaaa aaaggttttt gtgttacacc gcagtttagc gatcaggtta cccatgttgt 1740
tagcgaacag aatagctgta gcgaagttct gctgtggatt gaacgtcaga gtggtcagaa 1800
agttcagcct ggtggtgcag aaatgacacc gcatattctg gatattacct ggtttaccga 1860
aagcatgagc ctgggtaaac cggttaaagt tgaaccgcgt cattgtctgg gtgttagcga 1920
tagcagcgtt agccgtgata aagcaaccca agaaattccg gcatatggtt gtcagcgtcg 1980
tacaccgctg catcatcata ataaagaaat taccgatgcg ctggaaattc tggcactgag 2040
cgcaagcttt cagggtagcg aagcacgttt tctgggtttt acccgtgcaa gcagcgttct 2100
gaaaagcctg ccgtttcgtc tgcagagcgt tgaagaggtt aaagatctgc cgtggtgtgg 2160
tggtcatagc cagaccgtta ttcaagaaat cctggaagat ggtgtttgcc gtgaagttga 2220
aaccgtgaaa aatagcgaac atttccagag catgaaagca ctgaccagca tttttggtgt 2280
tggtattcgt accgcagata aatggtatcg tgatggtgtt cgtagcctga gcgatctgaa 2340
taatcttggt ggtaaactga ccgcagaaca gaaagcaggt ctgctgcatt acaccgatct 2400
gcagcagagc gtgacccgtg aagaagcagg caccgttgaa cagctgatta aaggtgcact 2460
gcagagcttt gtgccggatg tgcgtgttac catgaccggt ggttttcgtc gtggtaaaca 2520
agagggtcat gatgtggatt ttctgattac ccatcctgat gaagaagccc tgaacggcct 2580
gctgcgtaaa gcagttgcat ggctggatgg taaaggtagc gttctgtatt atcatgttcg 2640
tgcacgtagt cagaatttta gcggtagcaa taccatggat ggtcatgaaa cctgttatag 2700
cattattgca ctgccgaatg tttgtccgga aaaaccgagt ccggatgcag aaaaaattga 2760
accggatctg gataaaaaca gcctgcgtaa ttggaaagca gttcgtgttg atctggttgt 2820
ttgcccgtat agcgaatact tttatgcact gttaggttgg accggcagca aacattttga 2880
acgtgaactg cgtcgtttta gcctgcatgt gaaaaaaatg agcctgaata gccatggcct 2940
gtttgacatt cagaaaaagt gtcatcatcc ggcaaccagc gaagaagaaa tttttgcaca 3000
tctgggtctg ccgtatgttc cgcctagcga acgtaatgca taatgcacgt gaggatccaa 3060
ctcgagaact tagatggtat tagtgacctg taacagagca ttagcgcaag gtgatttttg 3120
tcttcttgcg ctaatttttt gtcatcaaac ctgtcgctag ttaagccagc cccgacaccc 3180
gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 3240
agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 3300
cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 3360
ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 3420
atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 3480
tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 3540
cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 3600
agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 3660
taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 3720
tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 3780
catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 3840
ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 3900
ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 3960
catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 4020
aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 4080
aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 4140
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 4200
atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 4260
gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 4320
tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 4380
ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 4440
gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 4500
agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 4560
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 4620
agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 4680
tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 4740
atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 4800
taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 4860
gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 4920
gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 4980
aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 5040
tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 5100
gtcagggggg cggagcctat ggaaa 5125
<210> 38
<211> 4909
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1142 expression vector full sequence
<400> 38
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcgaa cagcagaaac tgctggacac 1560
caaagagtat attatcaaac tggatgagct gaaaccggaa acgcgtaaag aaagtccggc 1620
aagcaaacag catattgaaa atctgcagaa acaagaaacc aaagagaaac tgattgcaga 1680
aagcagcacc ggtaatccga atgaacgtac catttttctg ctgaaccaga tggcagaaga 1740
acgtctgctg cagggtgaac attttaaagc aaaagcctat aagaacgcca ttaacgccct 1800
gaataatacc ggtgatttta tctcagatgc aaatgaagca ctgcgcctga aaggtattgg 1860
tgttagcgtg gcacagaaaa ttgaagaaat tgtgaaaacc aatacgctga gcagcctgaa 1920
tgaaatcaaa agcgataaag aacaccaggt gagcaaactg tttatgggta ttcatggtgt 1980
tggtccggtt agcgcaaaaa agtggtataa tgatggtctg cgtaccctgg aagatgttag 2040
ccagaaaccg gatctgacca gcaatcagac cctgggcctg aaatattacg atgaatggct 2100
ggaacgtatt ccgcgtgatg aatgtaccct gcataatgaa tttatgagcg atctggtgag 2160
ccagattgat ccgctggttc agtttaccat tggtggtagc tatcgtcgtg gtagcccgac 2220
ctgtggtgat gtggatttta tcattaccaa accgaatgcc gataacgaag agatgaaaga 2280
gattctggaa aagatcctgg tgaaaatcga acaggttggt tatctgaaat gtagcctgca 2340
gaaaaaacac agcaccaaat ttctgagcgg ttgtgcactg cctccgaatt atgcaagccg 2400
tctgccggaa tacagcgaag gtaaatgggg taaatgtcgt cgtattgatt ttctgatggt 2460
tccgtggaaa gaacgtggtg cagcatttat ctattttacc ggcaacgatt atttcaaccg 2520
tctgattcgt ctgaaagccg ttaaaaatgg tctggtgctg aatgaatcag gtctgtttaa 2580
acgcatcaaa tacgtgcagg gtaaaaacgt ggaagataaa accatgctga tcgaaagctt 2640
tagcgagaaa aaaatcttta agctgctggg cttcaaatat gttccgcctg aacagcgtaa 2700
ttttggtgca aataatccgc ctagcaaact gggtaaacat ctggatcagt ttcgcatcga 2760
tcacaaatat ttcgacaaag tggtgaaaga agagatcatt gacgacgatg ttatcgaggt 2820
ggattaatgc acgtgaggat ccaactcgag aacttagatg gtattagtga cctgtaacag 2880
agcattagcg caaggtgatt tttgtcttct tgcgctaatt ttttgtcatc aaacctgtcg 2940
ctagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3000
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3060
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3120
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3180
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3240
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3300
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3360
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3420
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3480
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 3540
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 3600
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 3660
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 3720
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 3780
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 3840
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 3900
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 3960
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4020
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4080
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4140
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4200
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4260
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4320
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4380
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4440
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4500
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4560
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4620
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4680
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 4740
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 4800
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 4860
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 4909
<210> 39
<211> 4768
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1108 expression vector full sequence
<400> 39
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagccgt accgattata gcgcaacccc 1560
gaatccgggt tttcagaaaa caccgcctct ggcagtgaaa aaaatcagcc agtatgcatg 1620
tcagcgtaaa accacactga ataactataa ccacatcttc accgatgcct ttgaaattct 1680
ggcagaaaac agcgaattca aagaaaacga agttagctac gtgaccttta tgcgtgcagc 1740
aagcgttctg aaaagcctgc cgtttaccat tattagcatg aaagataccg aaggtattcc 1800
gtgtctgggt gataaagtga aatgcatcat tgaagagatc atcgaagatg gtgaaagcag 1860
cgaagttaaa gcagttctga atgatgaacg ttaccagagc ttcaaactgt ttaccagcgt 1920
ttttggtgtt ggcctgaaaa ccagcgaaaa atggtttcgt atgggttttc gtagcctgag 1980
caaaatcatg agcgataaaa ccctgaaatt caccaaaatg cagaaagccg gtttcctgta 2040
ttatgaagat ctggtgagct gtgttacccg tgccgaagcc gaagcagttg gtgttctggt 2100
taaagaagca gtttgggcat ttctgccgga tgcatttgtt accatgaccg gtggttttcg 2160
tcgtggcaaa aaaatcggtc atgatgtgga ttttctgatt accagtccgg gtagcgcaga 2220
agatgaagaa cagctgctgc cgaaagttat taatctgtgg gaaaaaaaag gcctgctgct 2280
gtattacgat ctggttgaaa gcaccttcga gaaattcaaa ctgccgagcc gtcaggttga 2340
taccctggat cactttcaga aatgttttct tatcctgaag ctgcatcatc agcgtgttga 2400
tagcagcaaa agcaatcagc aagaaggtaa aacctggaaa gcaattcgtg ttgatctggt 2460
tatgtgcccg tatgaaaatc gtgcatttgc actgttaggt tggaccggta gtcgtcagtt 2520
tgaacgtgat attcgtcgtt atgcaaccca tgaacgtaaa atgatgctgg ataatcatgc 2580
cctgtacgat aaaacgaaac gcgtgttcct gaaagccgaa agcgaagaag aaatttttgc 2640
acatctgggc cttgattaca ttgaaccgtg ggaacgtaat gcctaatgca cgtgaggatc 2700
caactcgaga acttagatgg tattagtgac ctgtaacaga gcattagcgc aaggtgattt 2760
ttgtcttctt gcgctaattt tttgtcatca aacctgtcgc tagttaagcc agccccgaca 2820
cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 2880
acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 2940
acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 3000
aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 3060
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 3120
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 3180
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 3240
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 3300
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 3360
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 3420
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 3480
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 3540
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 3600
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 3660
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 3720
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 3780
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 3840
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 3900
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 3960
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 4020
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 4080
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 4140
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 4200
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 4260
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 4320
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 4380
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 4440
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 4500
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 4560
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 4620
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 4680
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 4740
ctcgtcaggg gggcggagcc tatggaaa 4768
<210> 40
<211> 5149
<212> DNA
<213> Artificial Sequence
<220>
<223> PP1075 expression vector full sequence
<400> 40
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 60
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 120
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 180
gaagatctcg atccgcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa 240
accctatgct actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg 300
acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg 360
gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg 420
acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg 480
tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc 540
gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg 600
tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac 660
tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc 720
tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa 780
tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag 840
ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg 900
cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca 960
cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg 1020
agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg 1080
ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc 1140
aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc 1200
aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta 1260
accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg 1320
acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg 1380
cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc 1440
tgacgctttt tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga 1500
ggaattaacc atgcatcatc atcaccatca cggcagcgat ccgctgcagg cagttcatct 1560
gggtccgcgt aaaaaacgtc cgcgtcagct gggtacaccg gttgcaagca ccccgtatga 1620
tattcgtttt cgtgatctgg ttctgttcat cctggaaaaa aagatgggta caacccgtcg 1680
tgcatttctg atggaactgg cacgtcgtaa aggttttcgt gttgaaaatg aactgagcga 1740
tagcgttacc catattgttg cagaaaataa cagcggtagt gatgttctgg aatggctgca 1800
actgcagaac attaaagcaa gcagcgaact ggaactgctg gatattagct ggctgattga 1860
atgtatgggt gcaggtaaac cggttgaaat gatgggtcgt catcagctgg ttgttaatcg 1920
taatagcagc ccgagtccgg ttccgggtag ccagaatgtt ccggcaccgg cagtgaaaaa 1980
aatcagtcag tatgcatgtc agcgtcgtac cacactgaat aactataatc agctgtttac 2040
cgatgcactg gatattctgg cagaaaatga tgagctgcgc gaaaatgaag gtagctgtct 2100
ggcatttatg cgtgccagca gcgttctgaa aagcctgccg tttccgatta ccagcatgaa 2160
agataccgaa ggtattccgt gtctgggtga taaagtgaaa agcattattg aaggcatcat 2220
cgaagatggc gaaagcagtg aagcaaaagc agttctgaat gatgaacgct acaaaagctt 2280
caaactgttt accagcgttt ttggtgttgg tctgaaaacc gcagaaaaat ggtttcgtat 2340
gggttttcgt accctgagca aaattcagag cgataaaagt ctgcgtttta cccagatgca 2400
gaaagcaggt tttctgtatt atgaagatct ggtgagctgc gttaatcgtc cggaagccga 2460
agcagttagc atgctggtta aagaagcagt tgttaccttt ctgccggatg cgctggttac 2520
catgaccggt ggttttcgtc gcggaaaaat gacaggtcat gatgtggatt ttctgattac 2580
ctcaccggaa gcaaccgaag atgaagaaca gcaactgctg cataaagtta ccgatttttg 2640
gaaacagcag ggtctgctgc tgtattgtga tatcctggaa tcaaccttcg agaaattcaa 2700
acagccgagc cgtaaagttg atgccctgga tcattttcag aagtgttttc tgatcctgaa 2760
actggatcat ggtcgtgttc atagcgaaaa aagcggtcag caagaaggta aaggttggaa 2820
agcaattcgt gtggatctgg ttatgtgtcc gtatgatcgt cgtgcctttg cactgttagg 2880
ttggaccggt agccgtcagt ttgaacgtga tctgcgtcgt tatgcaaccc atgaacgtaa 2940
aatgatgctg gataatcatg cactgtatga tcgcaccaaa cgtgtttttc tggaagcaga 3000
aagcgaagaa gaaatctttg cacatctggg ccttgattac attgaaccgt gggaacgtaa 3060
tgcataatgc acgtgaggat ccaactcgag aacttagatg gtattagtga cctgtaacag 3120
agcattagcg caaggtgatt tttgtcttct tgcgctaatt ttttgtcatc aaacctgtcg 3180
ctagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3240
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3300
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3360
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3420
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3480
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3540
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3600
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3660
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3720
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 3780
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 3840
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 3900
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 3960
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 4020
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 4080
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 4140
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 4200
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4260
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4320
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4380
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4440
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4500
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4560
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4620
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4680
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4740
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4800
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4860
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4920
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 4980
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 5040
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 5100
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 5149
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> PG1350 oligonucleotide
<400> 41
gcgtcacgct accaacca 18
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5858 oligonucleotide
<400> 42
gtcctcaatc gcactggaaa 20
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5859 oligonucleotide
<400> 43
gtcctcaatc gcactggaag 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5860 oligonucleotide
<400> 44
gtcctcaatc gcactggaac 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5861 oligonucleotide
<400> 45
gtcctcaatc gcactggaat 20
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5864 oligonucleotide
<400> 46
gtcctcaatc gcactggaat t 21
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5865 oligonucleotide
<400> 47
gtcctcaatc gcactggaat tg 22
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5866 oligonucleotide
<400> 48
gtcctcaatc gcactggaat tga 23
<210> 49
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5868 oligonucleotide
<400> 49
gtcctcaatc gcactggaag t 21
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5869 oligonucleotide
<400> 50
gtcctcaatc gcactggaag c 21
<210> 51
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5870 oligonucleotide
<400> 51
gtcctcaatc gcactggaaa catcaaggtc 30
<210> 52
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5871 oligonucleotide
<400> 52
gtcctcaatc gcactggaaa catcaaggtc atacggaacg 40
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5872 oligonucleotide
<400> 53
gtcctcaatc gcactggaat g 21
<210> 54
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> PG5867 oligonucleotide
<400> 54
gtcctcaatc gcactggaat tgac 24
Claims (22)
- 주형 독립적 핵산 합성(template independent nucleic acid synthesis)을 위한 서열 번호 26, 6, 28, 8, 21-25, 27, 1-5 및 7 중 어느 하나와 적어도 85% 동일성을 갖는 적어도 하나의 핵산 중합효소의 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 26 또는 6인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 1 또는 21인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 2 또는 22인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 3 또는 23인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 4 또는 24인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 5 또는 25인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 7 또는 27인 용도.
- 제1항에 있어서,
하나 이상의 핵산 중합효소가 서열 번호 8 또는 28인 용도.
- 제1항 내지 제11항 중 어느 한 항에 있어서,
서열 동일성이 적어도 90%인 용도.
- 제1항 내지 제12항 중 어느 한 항에 있어서,
서열 동일성이 적어도 95%인 용도.
- 제1항 내지 제13항 중 어느 한 항에 있어서,
서열 동일성이 98% 이상인 용도.
- 제1항 내지 제14항 중 어느 한 항에 있어서,
서열 동일성이 100%인 용도.
- 다음 단계를 포함하는 원하는 핵산을 합성하는 방법:
(a) 단일 용기(single vessel)에 적어도 하나의 핵산 기질, 과량의 유리 비차단 뉴클레오사이드 트리포스페이트(free unblocked nucleoside triphosphate) 및 서열 번호: 26, 6, 28, 8, 21-25, 27, 1-5 및 7 중 어느 하나와 적어도 85% 동일성을 갖는 적어도 하나의 주형 독립적 핵산 중합효소(template independent nucleic acid polymerase)를 혼합하는 단계;
(b) 새로운 핵산 분자를 형성하기 위하여 상기 적어도 하나의 주형 독립적 핵산 중합효소가 활성이고 반응에 존재하는 핵산 기질 분자에 단일 뉴클레오타이드만 첨가하는 조건 하에서 부분 (a)의 혼합물을 반응시키는 단계;
(c) 유리 뉴클레오타이드(free nucleotide) 및 상기 주형 독립적 핵산 중합효소로부터 새로운 핵산 분자를 분리하는 단계; 및
(d) 원하는 합성 핵산을 수득하기 위하여 단계 (a)-(c)를 반복하는 단계, 여기서 단계 (c)의 새로운 핵산 분자는 원하는 핵산이 합성될 때까지 단계 (a)의 핵산 기질로서 역할을 함.
- 제16항에 있어서,
상기 주형 독립적 핵산 중합효소의 서열 동일성이 적어도 90%인 방법.
- 제16항 또는 제17항에 있어서,
주형 독립적 핵산 중합효소의 서열 동일성이 적어도 95%인 방법.
- 제16항 내지 제18항 중 어느 한 항에 있어서,
98%인 방법.
- 제16항 내지 제19항 중 어느 한 항에 있어서,
주형 독립적 핵산 중합효소의 서열 동일성이 100%인 방법.
- 서열 번호 8과 적어도 85% 동일한 폴리펩티드를 코딩하는 핵산.
- 서열 번호 28과 적어도 85% 동일한 폴리펩티드를 코딩하는 핵산.
- 서열 번호 8과 적어도 85% 동일한 폴리펩티드.
- 서열 번호 28과 적어도 85% 동일한 폴리펩티드.
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US5808045A (en) | 1994-09-02 | 1998-09-15 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides |
US5763594A (en) | 1994-09-02 | 1998-06-09 | Andrew C. Hiatt | 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds |
DE10215035A1 (de) | 2002-04-05 | 2003-10-23 | Roche Diagnostics Gmbh | Rekombinante terminale Deoxynukleotidyltransferase mit verbesserter Funktionalität |
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