KR20170024129A - 항균제 - Google Patents
항균제 Download PDFInfo
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- KR20170024129A KR20170024129A KR1020177004886A KR20177004886A KR20170024129A KR 20170024129 A KR20170024129 A KR 20170024129A KR 1020177004886 A KR1020177004886 A KR 1020177004886A KR 20177004886 A KR20177004886 A KR 20177004886A KR 20170024129 A KR20170024129 A KR 20170024129A
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Abstract
본 발명은 그람-음성 박테리아에 대항하는 항균제, 특히 그람-음성 박테리아의 세포벽을 분해하는 활성을 갖는 효소 및 N- 또는 C-말단에서 효소에 융합된 펩티드 신장으로 구성된 융합 단백질에 관한 것이다. 게다가, 본 발명은 상기 융합 단백질을 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 벡터 및 상기 핵산 분자 또는 상기 벡터를 포함하는 숙주 세포에 관한 것이다. 또한, 본 발명은 특히 그람-음성 박테리아 감염의 치료 또는 예방을 위한 의약으로서, 진단 수단으로서 또는 화장품 성분으로서 사용하기 위한 상기 융합 단백질에 관한 것이다. 본 발명은 또한 식료품의, 식품 가공 장비의, 식품 가공 공장의, 식료품과 접촉하게 되는 표면의, 의료 장치의, 병원 및 수술 표면의 그람-음성 박테리아 오염의 치료 또는 예방에 관한 것이다. 게다가, 본 발명은 상기 융합 단백질을 포함하는 약학 조성물에 관한 것이다.
Description
본 발명은 그람-음성 박테리아에 대항하는 항균제, 특히 그람-음성 박테리아의 세포벽을 분해하는 활성을 갖는 효소 및 N- 또는 C-말단 상에서 효소에 융합된 부가적인 펩티드 신장으로 구성된 융합 단백질에 관한 것이다. 게다가, 본 발명은 상기 융합 단백질을 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 벡터 및 상기 핵산 분자 또는 상기 벡터를 포함하는 숙주 세포에 관한 것이다. 또한, 본 발명은 특히 그람-음성 박테리아 감염의 치료 또는 예방을 위한 의약으로서, 진단 수단으로서 또는 화장품 성분으로서 사용하기 위한 상기 융합 단백질에 관한 것이다. 본 발명은 또한 식료품의, 식품 가공 장비의, 식품 가공 공장의, 식료품과 접촉하게 되는 표면의, 의료 장치의, 병원 및 수술 표면의 그람-음성 박테리아 오염의 치료 또는 예방에 관한 것이다. 게다가, 본 발명은 상기 융합 단백질을 포함하는 약학 또는 화장 조성물에 관한 것이다.
그람-음성 박테리아는 홀마크로서 특징적인 비대칭 이중층을 갖는 외부 막을 소유한다. 외부 막 이중층은 인지질(주로 포스파티딜 에탄올아민)을 함유하는 내부 단층 및 단일 당지질인 리포폴리사카라이드(LPS)로 주로 구성된 외부 단층으로 이루어진다. 박테리아 계에는 엄청난 다양성의 LPS 구조가 있고, LPS 구조는 우세한 환경적 조건에 대한 반응으로 개질될 수 있다. LPS 층의 안정성 및 상이한 LPS 분자 사이의 상호작용은 주로, LPS 분자의 음이온 성분(지질 A 및 내부 코어 내의 포스페이트 기와 KDO의 카르복실기)과 2 가 이온(Mg2 +, Ca2 +)의 정전기적 상호작용에 의해 달성된다. 게다가, 불포화 지방산의 부재에 의해 선호되는 지질 A의 소수성 부분의 집약 및 정렬된 패킹은 고 점도를 갖는 단단한 구조를 형성한다. 이것은 친유성 분자를 투과하기 힘들게 하고, 외부 막(OM)에 대한 부가적인 안정성을 부여한다.
살균 또는 세균발육저지 작용을 갖는 다양한 유형의 작용제, 예를 들어, 항생제, 엔돌리신, 항균성 펩티드 및 디펜신이 알려져 있다. 그러나 미생물의 항생제에 대한 증가하는 내성은 박테리아에 의해 야기되는 감염을 더욱더 치료하기 어렵게 한다. 특히 어려운 점은 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 및 엔테로박테리아세아에(Enterobacteriaceae)와 같은 그람-음성 박테리아에 의해 야기되는 감염이다.
엔돌리신은 박테리오파지(또는 박테리아성 바이러스)에 의해 코딩되는 펩티도글리칸 가수분해효소이다. 이들은 상 증식의 용해 사이클 중 후기 유전자 발현 동안 합성되며, 박테리아성 펩티도글리칸의 분해를 통해 감염된 세포로부터 자손 비리온의 방출을 매개한다. 이들은 β(1,4)-글리코실라아제(라이소자임), 트랜스글리코실라아제, 아미다아제 또는 엔도펩티다아제이다. 엔돌리신의 항균성 적용은 이미 1991년에 Gasson(GB2243611)에 의해 제안되었다. 엔돌리신의 치사 능력이 오래전부터 알려져 있었음에도 불구하고, 항균제로의 상기 효소의 용도는 항생제의 성공 및 우월성으로 인해 무시되었다. 비로소 복합 항생제 내성 박테리아가 출연된 후에야, 엔돌리신으로 인간 병원체와 대항하는 상기 단순한 개념에 관심을 갖게 되었다. 완전히 새로운 종류의 항균제를 개발하려는 강력한 필요성이 부각되었고, '엔자바이오틱(enzybiotics)' - '효소'와 '항생제'의 혼성용어 - 로서 사용되는 엔돌리신은 상기 요구를 완벽하게 충족하였다. 2001년에, Fischetti 와 공동연구자는 최초로 그룹 A 스트렙토코시에 대한 박테리오파지 C1 엔돌리신의 치료 잠재력을 입증하였다(Nelson et al., 2001). 이후 많은 공개문헌이 엔돌리신을 박테리아 감염, 특히 그람-양성 박테리아에 의한 감염을 통제하는데 매력적이고 보완적인 대안으로서 성립하였다. 이어서 다른 그람-양성 병원체, 예컨대 스트렙토코쿠스 프네우모니아에(Streptococcus pneumoniae )(Loeffler et al., 2001), 바실러스 안트라시스(Bacillus anthracis)(Schuch et al., 2002), S. 아갈락티아에(S. agalactiae)(Cheng et al., 2005) 및 스타필로코쿠스 아우레우스(Staphylococcus aureus)(Rashel et al, 2007)에 대항하는 상이한 엔돌리신이 엔자바이오틱으로서의 효능이 입증되었다. 요즘, 엔돌리신 요법의 가장 중요한 도전과제는, 외부 막이 펩티도글리칸으로부터의 엔돌리신의 접근을 막고 있으므로, 엔돌리신의 외생 작용에 대한 그람-음성 박테리아의 무감각에 놓여있다. 이것은 현재 중요한 그람-음성 병원체에 대한 효과적인 엔돌리신의 범위의 팽창을 방해하고 있다.
항균성 펩티드(AMP)는 사실상 모든 유기체에서 발견될 수 있는 넓은 범위의 짧은, 양이온성 또는 양친매성 유전자-코딩 펩티드 항생제를 나타낸다. 상이한 AMP 는 상이한 특성을 나타내고, 상기 종류의 많은 펩티드는 항생제로서 뿐만 아니라 세포-투과성 펩티드에 대한 주형으로서 집중적으로 연구된다. 몇몇 공통 특징(예를 들어, 양이온성, 양친매성 및 짧은 크기)을 공유함에도 불구하고, AMP 서열은 크게 다르며, 관찰된 AMP 형태의 다양성을 도모하기 위해 4 개 이상의 구조적 그룹(α-나선형, β-판형, 확장형 및 루프형)이 제안되었다. 마찬가지로, 항생제로서 작용하는 여러 방식이 제안되었고, 예를 들어, 많은 상기 펩티드의 일차적 표적은 세포막인 반면, 다른 펩티드의 경우에는 일차적 표적은 세포질 침입 및 핵심 대사 기능의 파괴인 것이 제시되었다. AMP는 대부분의 AMP에 대한 경우에서와 같이, 예를 들어, 막 내 구멍을 형성함으로써, 특이적 표적 결합의 부재에도 불구하고 협동적인 활성을 나타내기에 충분히 농축될 수 있다. 그러나, 상기 현상은 오직 모델 인지질 이중층에서만 관찰되었고, 일부 경우에서, 6 개의 인지질 분자 당 1 개의 펩티드 분자 정도로 높았던 막 내 AMP 농도는 상기 사건이 일어나기를 필요로 하였다. 그렇지 않다면, 상기 농도는 전체 막 포화와 가깝다. AMP에 대한 최소 억제 농도(MIC)는 전형적으로 저 마이크로몰 범위에 있으므로, 상기 역치의 타당성 및 생체 내 이들의 중요성과 관련하여 당연하게도 회의론이 일어나고 있다(Melo et al., Nature reviews, Microbiology, 2009, 245).
디펜신은 척추동물 및 무척추동물 모두에서 발견되는 작은, 양이온성, 시스테인- 및 아르기닌-풍부 항균성 펩티드의 큰 계열이다. 디펜신은 시스테인의 간격 패턴에 따라, 식물, 무척추동물, α-, β- 및 θ-디펜신의 5 개의 그룹으로 나뉜다. 마지막 3 개는 대부분 포유동물에서 발견된다. α-디펜신은 호중구 및 장 상피에서 발견되는 단백질이다. β-디펜신은 가장 널리 분포되어 있고, 백혈구 및 많은 종류의 상피 세포에 의해 분비된다. θ-디펜신은 예를 들어, 히말라야 원숭이(rhesus macaque)의 백혈구를 제외하고는 좀처럼 발견되지 않는다. 디펜신은 박테리아, 진균 및 많은 외피 및 비-외피 바이러스에 대항하여 활성이다. 그러나, 박테리아의 효과적인 치사에 필요한 농도는 대부분 높다, 즉 μ-몰 범위이다. 많은 펩티드의 활성은 생리학적 염 조건, 2가 양이온 및 혈청의 존재에 제한될 수 있다. 소수성 아미노산 잔기의 함량에 따라 디펜신은 또한 용혈 활성을 보인다.
따라서, 신규한 항균제에 대한 필요성이 있다.
상기 목적은 청구항에 정의된 주제에 의해 해결된다.
본원에서 사용되는 "단백질"이라는 용어는 "폴리펩티드"라는 용어와 동의어로 언급된다. 본원에서 사용되는 "단백질"이라는 용어는 특이적 서열 내 펩티드 결합에 의해 연결된 아미노산 잔기의 선형 중합체를 말한다. 단백질의 아미노산 잔기는 예를 들어, 카보히드레이트 및 포스페이트와 같은 다양한 기의 공유 부착에 의해 개질될 수 있다. 다른 성분은 헴 또는 지질과 같이 폴리펩티드 사슬과 좀더 느슨하게 연합되어서, 본원에서 "단백질"이라는 용어에 의해 또한 포함되는 공액 단백질이라는 것을 산출한다. 폴리펩티드 사슬이 폴딩되는 다양한 방식이 특히 알파 나선 및 베타-병풍구조의 존재와 관련하여 설명된다. 본원에서 사용되는 "단백질"이라는 용어는 모든-알파, 모든-베타, 알파/베타 및 알파 + 베타인 모든 4 가지 종류의 단백질을 말한다. 게다가, "단백질"이라는 용어는 복합체를 말하며, 상기 복합체는 동종체(homomer)를 말한다.
본원에서 사용되는 "융합 단백질"이라는 용어는 2 가지 핵산 서열의 융합으로부터 수득되는 발현 생성물을 말한다. 이러한 단백질은 예를 들어, 재조합 DNA 발현 시스템에서 생성될 수 있다. 게다가, 본원에서 사용되는 "융합 단백질"이라는 용어는 예를 들어, 효소로서 제1 아미노산 서열의, 제2 또는 추가의 아미노산 서열과의 융합을 말한다. 제2 또는 추가의 아미노산 서열은 도메인 또는 임의의 종류의 펩티드 신장을 정의할 수 있다. 바람직하게는, 상기 제2 및/또는 추가의 아미노산 서열은 외래의 것이고 제1 아미노산 서열의 임의의 도메인과 실질적으로 상동은 아니다.
본원에서 사용되는 "펩티드 신장"이라는 용어는 효소와 같은 단백질에 연결된 임의의 종류의 펩티드를 말한다.
본원에서 사용되는 "펩티드"라는 용어는 약 2 내지 약 100 개의 아미노산 잔기, 더욱 바람직하게는 약 4 내지 약 50 개의 아미노산 잔기, 더욱 바람직하게는 약 5 내지 30 개의 아미노산 잔기로 이루어지는 짧은 폴리펩티드를 말하고, 하나의 아미노산 잔기의 아미노기는 펩티드 결합에 의해 또다른 아미노산 잔기의 카르복실기에 연결된다. 펩티드는 특이적인 작용을 가질 수 있다. 펩티드는 자연 발생적 펩티드이거나 합성적으로 디자인되고 생성된 펩티드일 수 있다. 펩티드는 예를 들어, 효소적 또는 화학적 분할에 의해 고유의 단백질로부터 유래 또는 제거될 수 있거나, 또는 통상적인 펩티드 합성 기술(예를 들어, 고상 합성) 또는 분자 생물학 기술(참조, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N. Y.(1989))을 사용하여 제조될 수 있다. 자연 발생적 펩티드의 예는 항균성 펩티드, 디펜신, 스시 펩티드이다. 합성적으로 생성된 펩티드의 예는 폴리양이온성, 양친매성 또는 소수성 펩티드이다. 본 발명의 의미에서 펩티드는 His-태그, Strep-태그, 티오레독신 또는 말토오스 결합 단백질(MBP) 등을 말하지는 않으며, 이들은 단백질을 정제하거나 위치화시키기 위해 사용된다.
본원에서 사용되는 "엔돌리신"이라는 용어는 박테리아 세포벽을 가수분해하는데 적합한 효소를 말한다. "엔돌리신"은 하기 활성 중 하나 이상을 갖는 하나 이상의 "효소적으로 활성인 도메인"(EAD)을 포함한다: 엔도펩티다아제, 키티나아제, T4 유사 뮤라미니다아제, 람다 유사 뮤라미니다아제, N-아세틸-뮤라모일-L-알라닌-아미다아제(아미다아제), 뮤라모일-L-알라닌-아미다아제, 뮤라미다아제, 용해성 트랜스글리코실라아제(C), 용해성 트랜스글리코실라아제(M), N-아세틸-뮤라미다아제, N-아세틸-글루코사미니다아제(라이소자임) 또는 예를 들어 KZ144 및 EL188 과 같은 트랜스글리코실라아제. 부가적으로, 엔돌리신은 또한 효소적으로 비활성이고, 숙주 박테리아의 세포벽에 결합할 수 있는 영역(소위 CBD(세포벽 결합 도메인))을 함유할 수 있다.
본원에서 사용되는 "EAD"라는 용어는 엔돌리신의 효소적으로 활성인 도메인을 말한다. EAD는 박테리아성 펩티도글리칸을 가수분해하는 것을 담당한다. 이것은 엔돌리신의 하나 이상의 효소적 활성을 나타낸다. EAD는 또한 1 개 초과의 효소적으로 활성인 모듈로 구성될 수 있다.
"오토리신"이라는 용어는 엔돌리신과 관련되나, 박테리아에 의해 코딩되고, 예를 들어, 세포 분열에 관여하는 효소를 말한다. 오토리신에 대한 개괄적인 내용은 문헌 "Bacterial peptidoglycan(murein)hydrolases. Vollmer W, Joris B, Charlier P, Foster S. FEMS Microbiol Rev. 2008 Mar;32(2):259-86"에서 찾을 수 있다.
본원에서 사용되는 "박테리오신"이라는 용어는 다른 박테리아의 성장을 억제할 수 있는 단백질-유사, 폴리펩티드-유사 또는 펩티드-유사 성분을 말한다. 바람직하게는 상기 억제는 박테리오신의 특이적 수용체에 대한 상기 다른 박테리아의 흡수라는 방식에 의해 특이적으로 이루어진다. 일반적으로, 박테리오신은 미생물에 의해 생성된다. 그러나, 본원에서 사용되는 "박테리오신"이라는 용어는 미생물에 의해 생성된 단리된 형태 또는 합성적으로 생성된 형태 모두를 말하며, 또한 이들의 모 박테리오신의 활성을 실질적으로 보유하나, 이들의 서열은 하나 이상의 아미노산 잔기의 삽입 또는 결실에 의해 변경된 변이체를 말한다.
본원에서 사용되는 "항균성 펩티드"(AMP)라는 용어는 살균 및/또는 세균발육저지 활성을 갖는 임의의 펩티드를 말한다. 따라서, 본원에서 사용되는 "항균성 펩티드"라는 용어는 특히, 항-박테리아, 항-진균, 항-사상균, 항-구충, 항-원생동물, 항-바이러스, 항-감염성, 항-전염 및/또는 살균, 살조류, 살아메바, 살미생물, 살박테리아, 살진균, 살구충, 치사원생동물(protozoacidal), 살원생동물 특성을 갖는 임의의 펩티드를 말한다.
본원에서 사용되는 "디펜신"이라는 용어는 동물, 바람직하게는 포유동물, 더욱 바람직하게는 인간 내에 존재하는 펩티드를 말하며, 상기 디펜신은 감염성 박테리아 및/또는 감염성 바이러스 및/또는 진균과 같은 외부 성분의 파괴와 같은 선천적 숙주 방어 시스템에서 역할을 한다. 디펜신은 비-항체 살균 및/또는 살종양 단백질, 펩티드 또는 폴리펩티드이다. "디펜신"에 대한 예는 "포유동물 디펜신,"알파-디펜신, 베타-디펜신, 인돌리시딘 및 마가이닌이다. 본원에서 사용되는 "디펜신"은 동물 세포로부터 단리된 형태 또는 합성적으로 생성된 형태를 모두 말하며, 또한 이들의 모 단백질의 세포독성 활성을 실질적으로 보유하나, 이들의 서열은 하나 이상의 아미노산 잔기의 삽입 또는 결실에 의해 변경된 변이체를 말한다.
*본원에서 사용되는 "스시 펩티드"라는 용어는 짧은 공통 반복을 갖는 상보적 조절 단백질(CCP)을 말한다. 스시 펩티드의 스시 모듈은 많은 상이한 단백질에서 단백질-단백질 상호작용 도메인으로서 기능한다. 스시 도메인을 함유하는 펩티드는 항균 활성을 갖는다고 제시된다.
본원에서 사용되는 "양이온성 펩티드"라는 용어는 양전하 아미노산 잔기를 갖는 펩티드를 말한다. 바람직하게는 양이온성 펩티드는 9.0 이상의 pKa-값을 갖는다. 전형적으로는, 양이온성 펩티드의 아미노산 잔기 중 4 개 이상은 양전하, 예를 들어, 리신 또는 아르기닌일 수 있다. "양전하"는 약 생리학적 조건에서 네트 양전하를 갖는 아미노산 잔기의 측쇄를 말한다. 재조합으로 생성될 수 있는 자연 발생적 양이온성 펩티드의 예는 디펜신, 마가이닌, 멜리틴, 및 세크로핀이다.
본원에서 사용되는 "폴리양이온성 펩티드"라는 용어는 대부분 리신 및/또는 아르기닌 잔기로 구성된 합성적으로 생성된 펩티드를 말한다.
본원에서 사용되는 "양친매성 펩티드"라는 용어는 친수성 및 소수성 작용기를 모두 갖는 펩티드를 말한다. 바람직하게는, 본원에서 사용되는 "양친매성 펩티드"라는 용어는 친수성 및 소수성 기의 정의된 배열을 갖는 펩티드를 말한다, 예를 들어 양친매성 펩티드는 예를 들어, 나선의 한 면을 따라 우세한 비-극성 측쇄를, 표면의 나머지를 따라 극성 잔기를 갖는 알파 나선일 수 있다.
본원에서 사용되는 "소수성 기"라는 용어는 실질적으로 수 불용성이나 유상에는 가용성인, 물에 또는 수성상에서보다 유상에서의 용해도가 높은 아미노산 측쇄와 같은 화학적 기를 말한다. 물에서, 소수성 측쇄를 갖는 아미노산은 비수성 환경을 생성하기 위해 서로 상호작용한다. 소수성 측쇄를 갖는 아미노산의 예는 알라닌, 발린, 류신, 이소류신, 페닐알라닌, 히스티딘, 트립토판 및 티로신이다.
본원에서 사용되는 "결실"이라는 용어는 각각의 출발 서열로부터 1, 2, 3, 4, 5 개 이상의 아미노산 잔기의 제거를 말한다.
본원에서 사용되는 "삽입"또는 "부가"라는 용어는 각각의 출발 서열로부터 1, 2, 3, 4, 5 개 이상의 아미노산 잔기의 삽입 또는 부가를 말한다.
본원에서 사용되는 "치환"이라는 용어는 특정 위치에 위치하는 아미노산 잔기의 상이한 잔기에 대한 교환을 말한다.
본 발명은 그람-음성 박테리아에 대항하는 신규 항균제, 특히 그람-음성 박테리아의 세포벽을 분해하는 활성을 갖는 효소 및 N- 또는 C-말단 또는 양 말단 상에서 효소에 융합된 부가적인 펩티드 신장으로 구성된 융합 단백질에 관한 것이다.
본 발명의 하나의 양상에서, 그람-음성 박테리아의 세포벽을 분해하는 활성을 갖는 효소는 엔돌리신, 오토리신 또는 박테리오신이다.
본 발명의 또다른 양상에서, 효소는 또한 효소적으로 비활성이고, 숙주 박테리아의 세포벽에 결합할 수 있는 영역(소위 CBD(세포벽 결합 도메인))을 함유할 수 있다.
본 발명에 따른 바람직한 융합 단백질은 SEQ ID NO: 36 내지 63 에 열거되어 있다. SEQ ID NO:36 내지 63에 따른 융합 단백질은 N-말단 상에 하나 이상의 부가적인 아미노산 잔기를 포함할 수 있다. 바람직하게는 부가적인 아미노산 잔기는 메티오닌이다.
바람직하게는, 엔돌리신은, 엔테로박테리아세아에(Enterobacteriaceae)(에스케리챠(Escherichia), 특히 E. 콜라이(E. coli), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 특히 K. 프네우모니아에(K. pneumoniae ), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia), 예르시니아(Yersinia)), 슈도모나다세아에(Pseudomonadaceae)(슈도모나스(Pseudomonas), 특히 P. 아에루기노사(P. aeruginosa), 부르크홀데리아(Burkholderia), 스테노트로포모나스(Stenotrophomonas), 쉐와넬라(Shewanella), 스핑고모나스(Sphingomonas), 코마모나스(Comamonas)), 네이세리아(Neisseria), 모라셀라(Moraxella), 비브리오(Vibrio), 아에로모나스(Aeromonas), 브루셀라(Brucella), 프란시셀라(Francisella), 보르데텔라(Bordetella), 레지오넬라(Legionella), 바르토넬라(Bartonella), 콕시엘라(Coxiella), 헤모필러스(Haemophilus), 파스퉤렐라(Pasteurella), 만헤이미아(Mannheimia), 악티노바실러스(Actinobacillus), 가르드네렐라(Gardnerella), 스피로카에타세아에(Spirochaetaceae)(트레포네마(Treponema) 및 보렐리아(Borrelia)), 렙토스피라세아에(Leptospiraceae), 캄필로박테르(Campylobacter), 헬리코박테르(Helicobacter), 스필릴룸(Spirillum), 스트렙토바실러스(Streptobacillus), 박테로이다세아에(Bacteroidaceae)(박테로이데스(Bacteroides), 푸소박테리움(Fusobacterium), 프레보텔라(Prevotella), 포르피로모나스(Porphyromonas)), 아시네토박테르(Acinetobacter), 특히 A. 바우마니이(A. baumanii)와 같은 인간 또는 동물에 대해 병원성인 균주를 포함하는 박테리아 그룹, 과, 속 또는 종의 그람-음성 박테리아와 같은 그람-음성 박테리아에 대해 특이적인 박테리오파지에 의해 코딩된다.
*바람직하게는, 엔돌리신은, 엔테로박테리아세아에(Enterobacteriaceae)(에스케리챠(Escherichia), 특히 E. 콜라이(E. coli), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 특히 K. 프네우모니아에(K. pneumoniae ), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia), 예르시니아(Yersinia)), 슈도모나다세아에(Pseudomonadaceae)(슈도모나스(Pseudomonas), 특히 P. 아에루기노사(P. aeruginosa), 부르크홀데리아(Burkholderia), 스테노트로포모나스(Stenotrophomonas), 쉐와넬라(Shewanella), 스핑고모나스(Sphingomonas), 코마모나스(Comamonas)), 네이세리아(Neisseria), 모라셀라(Moraxella), 비브리오(Vibrio), 아에로모나스(Aeromonas), 브루셀라(Brucella), 프란시셀라(Francisella), 보르데텔라(Bordetella), 레지오넬라(Legionella), 바르토넬라(Bartonella), 콕시엘라(Coxiella), 헤모필러스(Haemophilus), 파스퉤렐라(Pasteurella), 만헤이미아(Mannheimia), 악티노바실러스(Actinobacillus), 가르드네렐라(Gardnerella), 스피로카에타세아에(Spirochaetaceae)(트레포네마(Treponema) 및 보렐리아(Borrelia)), 렙토스피라세아에(Leptospiraceae), 캄필로박테르(Campylobacter), 헬리코박테르(Helicobacter), 스필릴룸(Spirillum), 스트렙토바실러스(Streptobacillus), 박테로이다세아에(Bacteroidaceae)(박테로이데스(Bacteroides), 푸소박테리움(Fusobacterium), 프레보텔라(Prevotella), 포르피로모나스(Porphyromonas)), 아시네토박테르(Acinetobacter), 특히 A. 바우마니이(A. baumanii)와 같은 인간 또는 동물에 대해 병원성인 균주를 포함하는 박테리아 그룹, 과, 속 또는 종의 그람-음성 박테리아와 같은 그람-음성 박테리아에 의해 코딩된다.
박테리오신은 바람직하게는 상기 열거된 바와 같은 그람-음성 박테리아에 특이적이지만, 또한 덜 특이적일 수 있다.
본 발명에 따른 효소는, 엔테로박테리아세아에(Enterobacteriaceae)(에스케리챠(Escherichia), 특히 E. 콜라이(E. coli), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 특히 K. 프네우모니아에(K. pneumoniae ), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia), 예르시니아(Yersinia)), 슈도모나다세아에(Pseudomonadaceae)(슈도모나스(Pseudomonas), 특히 P. 아에루기노사(P. aeruginosa), 부르크홀데리아(Burkholderia), 스테노트로포모나스(Stenotrophomonas), 쉐와넬라(Shewanella), 스핑고모나스(Sphingomonas), 코마모나스(Comamonas)), 네이세리아(Neisseria), 모라셀라(Moraxella), 비브리오(Vibrio), 아에로모나스(Aeromonas), 브루셀라(Brucella), 프란시셀라(Francisella), 보르데텔라(Bordetella), 레지오넬라(Legionella), 바르토넬라(Bartonella), 콕시엘라(Coxiella), 헤모필러스(Haemophilus), 파스퉤렐라(Pasteurella), 만헤이미아(Mannheimia), 악티노바실러스(Actinobacillus), 가르드네렐라(Gardnerella), 스피로카에타세아에(Spirochaetaceae)(트레포네마(Treponema) 및 보렐리아(Borrelia)), 렙토스피라세아에(Leptospiraceae), 캄필로박테르(Campylobacter), 헬리코박테르(Helicobacter), 스필릴룸(Spirillum), 스트렙토바실러스(Streptobacillus), 박테로이다세아에(Bacteroidaceae)(박테로이데스(Bacteroides), 푸소박테리움(Fusobacterium), 프레보텔라(Prevotella), 포르피로모나스(Porphyromonas)), 아시네토박테르(Acinetobacter), 특히 A. 바우마니이(A. baumanii)와 같은 인간 또는 동물에 대해 병원성인 균주를 포함하는 박테리아 그룹, 과, 속 또는 종의 그람-음성 박테리아에 대항하여 세포벽 분해 활성을 갖는다.
파지로부터 유래된 또는 야생형 엔돌리신인 엔돌리신 부분의 구체적인 예는 하기 표에 열거된다:
또한 바람직한 것은 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 파지 ΦKZ 및 EL의 엔돌리신, 슈도모나스 퓨티다(Pseudomonas putida ) 파지의 엔돌리신, E. 콜라이(E. coli ) 파지 N4의 엔돌리신, 파지 LUZ24의 엔돌리신, gp61 뮤라미다아제, STM0016 엔돌리신 및 PSP3 엔돌리신 유래의 엔돌리신 부분이다.
엔돌리신 부분에 대한 추가의 예는 SEQ ID NO:1에 따른 phiKZgp144, SEQ ID NO:2에 따른 ELgp188, SEQ ID NO:3에 따른 살모넬라(Salmonella) 엔돌리신, SEQ ID NO:4에 따른 엔테로박테리아 파지 T4 엔돌리신, SEQ ID NO:5에 따른 아시네토박테르 바우마니이(Acinetobacter baumanii ) 엔돌리신, SEQ ID NO:18에 따른 E. 콜라이(E. coli ) 파지 K1F 엔돌리신, SEQ ID NO:34에 따른 OBPgpLYS, SEQ ID NO:20에 따른 PSP3 살모넬라(Salmonella) 엔돌리신(PSP3gp10), SEQ ID NO:21에 따른 E. 콜라이(E. coli ) 파지 P2 엔돌리신(P2gp09), SEQ ID NO:22에 따른 살모넬라 티피뮤리움(Salmonella typhimurium ) 파지 뮤라미다아제 STM0016, SEQ ID NO:23에 따른 E. 콜라이(E. coli ) 파지 N4 뮤라미다아제 N4-gp61 및 SEQ ID NO:24에 따른 N4-gp61 trunc., SEQ ID NO:25에 따른 KZ144로 이루어진 군으로부터 선택된다.
본 발명의 또다른 바람직한 구현예에서, 본 발명에 따른 융합 단백질의 엔돌리신, 오토리신 및 박테리오신은 아미노산 서열의 개질 및/또는 변형을 포함한다. 상기 변형 및/또는 개질은 결실, 삽입 및 부가, 치환 또는 이의 조합 및/또는 아미노산 잔기의 화학적 변화, 예를 들어, 비오티닐화, 아세틸화, 페길화, 아미노-, SH- 또는 카르복실-기의 화학적 변화와 같은 돌연변이를 포함할 수 있다. 본 발명에 따른 융합 단백질의 상기 엔돌리신, 오토리신 및 박테리오신은 각각의 야생형 엔돌리신, 오토리신 및 박테리오신의 용해성 활성을 나타낸다. 그러나, 상기 활성은 각각의 야생형 엔돌리신의 활성과 동일하거나, 그보다 높거나 낮을 수 있다. 상기 활성은 각각의 야생형 엔돌리신의 활성의 약 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 또는 약 200% 또는 심지어 그 이상일 수 있다. 활성은 예를 들어, 문헌 [Briers et al, J. Biochem . Biophys Methods 70: 531 - 533,( 2007)] 또는 [Donovan DM, Lardeo M, Foster- Frey J. FEMS Microbiol Lett . 2006 Dec ; 265(1)] 또는 유사한 공개문헌에 기재된 바와 같은 예를 들어, 플레이트 용해 어세이 또는 액체 용해 어세이와 같은 당업자에게 잘 알려진 어세이에 의해 측정될 수 있다.
바람직하게는, 본 발명에 따른 융합 단백질의 펩티드 신장은 엔돌리신, 오토리신 및 박테리오신의 N-말단 및/또는 C-말단에 융합된다. 특히 바람직한 구현예에서 상기 펩티드 신장은 효소의 N-말단에만 융합된다. 또다른 바람직한 구현예에서 펩티드 신장은 효소의 C-말단에만 융합된다. 그러나, 또한 바람직한 것은 N-말단 및 C-말단 상 모두에서 펩티드 신장을 갖는 개질된 융합 단백질이다. N-말단 및 C-말단 상의 상기 펩티드 신장은 동일 또는 구별되는 펩티드 신장일 수 있다. 펩티드 신장은 예를 들어, 클로닝 이유로 인해, 부가적인 아미노산 잔기에 의해 효소에 연결될 수 있다. 바람직하게는, 상기 펩티드 신장은 1, 2, 3, 4, 5, 6, 7, 8, 9 또는 10 개 이상의 부가적인 아미노산 잔기에 의해 융합 단백질에 연결될 수 있다. 바람직한 구현예에서 펩티드 신장은 부가적인 아미노산 잔기 글리신 및 세린(Gly-Ser) 또는 류신 및 글루탐산(Leu-Glu)에 의해 효소에 연결된다. 게다가, 본 발명에 따른 융합 단백질의 펩티드 신장은 N-말단 상에 부가적인 아미노산을 추가로 포함한다. 바람직하게는, 펩티드 신장은 아미노산 메티오닌(Met), 알라닌 및 메티오닌 및 글리신(Ala-Met-Gly-Ser) 또는 알라닌 및 메티오닌 및 글리신 및 세린(Ala-Met-Gly-Ser)을 포함한다.
본 발명에 따른 융합 단백질의 펩티드 신장은 바람직하게는 효소에 공유 결합된다. 바람직하게는, 상기 펩티드 신장은 5 개 이상, 더욱 바람직하게는 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 개 이상 또는 100 개 이상의 아미노산 잔기로 이루어진다. 특히 바람직한 것은 약 5 내지 약 100 개의 아미노산 잔기, 약 5 내지 약 50 개의 또는 약 5 내지 약 30 개의 아미노산 잔기를 포함하는 펩티드 신장이다. 더욱 바람직한 것은 약 6 내지 약 42 개의 아미노산 잔기, 약 6 내지 약 39 개의 아미노산 잔기, 약 6 내지 약 38 개의 아미노산 잔기, 약 6 내지 약 31 개의 아미노산 잔기, 약 6 내지 약 25 개의 아미노산 잔기, 약 6 내지 약 24 개의 아미노산 잔기, 약 6 내지 약 22 개의 아미노산 잔기, 약 6 내지 약 21 개의 아미노산 잔기, 약 6 내지 약 20 개의 아미노산 잔기, 약 6 내지 약 19 개의 아미노산 잔기, 약 6 내지 약 16 개의 아미노산 잔기, 약 6 내지 약 14 개의 아미노산 잔기, 약 6 내지 약 12 개의 아미노산 잔기, 약 6 내지 약 10 개의 아미노산 잔기 또는 약 6 내지 약 9 개의 아미노산 잔기를 포함하는 펩티드 신장이다.
바람직하게는, 펩티드 신장은 His-태그, Strep-태그, Avi-태그, Myc-태그, Gst-태그, JS-태그, 시스테인-태그, FLAG-태그 또는 당업계에 알려진 기타 태그와 같은 태그가 아니고, 티오레독신 또는 말토오스 결합 단백질(MBP)이 아니다. 그러나, 본 발명에 따른 펩티드 신장 및/또는 엔돌리신, 오토리신 또는 박테리오신은 그러한 태그 또는 태그들을 부가적으로 포함할 수 있다.
더욱 바람직하게는 펩티드 신장은 외부 막을 통해 융합 단백질을 인도하는 기능을 갖지만, 효소에 융합되지 않고 투여되었을 때 활성을 가질 수 있거나, 활성이 없거나 오직 적을 수 있다. 그람-음성 박테리아의 외부 막을 통해 융합 단백질을 인도하는 기능은 상기 펩티드 신장의 활성을 파괴하거나 투과하거나 탈안정화시키는 외부 막 또는 LPS 의 잠재력에 의해 야기된다.
본 발명의 추가의 양상에서 융합된 펩티드 신장은 발린, 이소류신, 류신, 메티오닌, 페닐알라닌, 트립토판, 시스테인, 알라닌, 티로신, 히스티딘, 트레오닌, 세린, 프롤린 및/또는 글리신의 하나 이상의 소수성 아미노산 잔기와 조합된, 리신, 아르기닌 및/또는 히스티딘의 하나 이상의 양전하 아미노산 잔기를 포함하는 양친매성 펩티드이다. 아미노산 잔기의 측쇄는 양이온성 및 소수성 표면이 펩티드의 반대 면에서 클러스팅 되도록 배향된다. 바람직하게는, 상기 펩티드 내 아미노산의 약 30, 40, 50, 60 또는 70% 초과는 양전하 아미노산이다. 바람직하게는, 상기 펩티드 내 아미노산 잔기의 약 30, 40, 50, 60 또는 70% 초과는 소수성 아미노산 잔기이다. 유리하게는, 양친매성 펩티드는 세포벽 분해 활성을 갖는 효소의 N-말단 및/또는 C-말단 말미에 융합되므로, 상기 단백질의 양친매성을 향상시킨다.
본 발명의 또다른 구현예에서, 효소에 융합된 양친매성 펩티드는 5 개 이상, 더욱 바람직하게는 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 또는 50 개 이상의 아미노산 잔기로 이루어진다. 바람직한 구현예에서 양친매성 펩티드의 상기 아미노산 잔기의 약 30, 40, 50, 60 또는 70% 이상은 아르기닌 또는 리신 잔기이고, 및/또는 양친매성 펩티드의 상기 아미노산 잔기의 약 30, 40, 50, 60 또는 70% 이상은 소수성 아미노산 발린, 이소류신, 류신, 메티오닌, 페닐알라닌, 트립토판, 시스테인, 알라닌, 티로신, 히스티딘, 트레오닌, 세린, 프롤린 및/또는 글리신이다.
바람직한 양친매성 펩티드는 SEQ ID NO:6에 따른 플레우로시딘(Pleurocidin), SEQ ID NO:7에 따른 세크로핀 P1, SEQ ID NO:8에 따른 부포린 II(Buforin II), SEQ ID NO:19에 따른 부포린 I 및 SEQ ID NO:9에 따른 마가이닌이다. 추가의 바람직한 양친매성 펩티드는 카테리디신(Cathelidicine)예를 들어, SEQ ID NO:10에 따른 LL-37, SEQ ID NO:26에 따른 니그로신 2(Nigrocine 2) 및 SEQ ID NO:27에 따른 아스카핀 5(Ascaphine 5)이다.
본 발명의 추가의 양상에서 융합된 펩티드 신장은 양성 네트 전하 및 약 50% 의 소수성 아미노산을 포함하는 항균성 펩티드이다. 항균성 펩티드는 약 12 내지 약 50 개의 아미노산 잔기의 길이를 갖는 양친매성이다.
본 발명에 따른 항균성 펩티드의 구체적인 예는 하기 표에 나열되어 있다.
본 발명의 추가의 양상에서 융합된 펩티드 신장은 문헌 [Ding JL, Li P, Ho B Cell Mol Life Sci. 2008 Apr;65(7-8):1202-19. The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria] 에 기재되어 있는 스시 펩티드이다. 특히 바람직한 것은 SEQ ID NO:32에 따른 스시 1 펩티드이다.
바람직한 스시 펩티드는 스시 펩티드 S1 및 S3 및 이의 다중체이다; FASEB J. 2000 Sep;14(12):1801-13.
본 발명의 추가의 양상에서 융합된 펩티드 신장은 디펜신, 바람직하게는 카테리디신, 세크로핀 P1, 세크로핀 A 또는 마가이닌 II 이다.
본 발명의 추가의 양상에서 융합된 펩티드 신장은 소수성 펩티드 예를 들어, SEQ ID NO: 28에 따른 아미노산 서열을 갖는 아피다에신(Apidaecine), SEQ ID NO:33에 따른 아미노산 서열을 갖는 WLBU2-변이체 및 SEQ ID NO:35에 따른 아미노산 서열을 갖는 월마흐1(Walmagh1)이다. 아미노산 서열 Phe-Phe-Val-Ala-Pro(SEQ ID NO:17)를 갖는 소수성 펩티드는 본 발명의 일부가 아니다.
본 발명의 또다른 바람직한 구현예에서 본 발명에 따른 융합 단백질의 펩티드 신장은 아미노산 서열의 개질 및/또는 변형을 포함한다. 상기 변형 및/또는 개질은 결실, 삽입 및 부가, 치환 또는 이의 조합 및/또는 아미노산 잔기의 화학적 변화, 예를 들어, 비오티닐화, 아세틸화, 페길화, 아미노-, SH- 또는 카르복실-기의 화학적 변화와 같은 돌연변이를 포함할 수 있다
본 발명에 따른 융합 단백질의 구체적인 예는 하기 표에 나열되어 있다.
본 발명에 따른 융합 단백질, 및 그러므로 특히 SEQ ID NO: 36 내지 63에 따른 특히 바람직한 융합 단백질은 N-말단 상에 메티오닌을 부가적으로 포함할 수 있다.
본 발명에 따른 융합 단백질, 및 그러므로 특히 SEQ ID NO: 36 내지 63에 따른 특히 바람직한 융합 단백질은 예를 들어, 정제를 위한 태그를 부가적으로 포함할 수 있다. 바람직한 것은 바람직하게는 융합 단백질의 C-말단 및/또는 N-말단에 있는 His6-태그이다. 상기 태그는 예를 들어, 클로닝 이유로 인해 부가적인 아미노산 잔기에 의해 융합 단백질에 연결될 수 있다. 바람직하게는 상기 태그는 1, 2, 3, 4, 5, 6, 7, 8, 9 또는 10 개 이상의 부가적인 아미노산 잔기에 의해 융합 단백질에 연결될 수 있다. 바람직한 구현예에서 융합 단백질은 부가적인 아미노산 잔기 리신 및 글리신(Lys-Gly) 또는 류신 및 글루탐산(Leu-Glu)에 의해 융합 단백질에 연결된 C-말단에서 His6-태그를 포함한다. 또다른 바람직한 구현예에서 융합 단백질은 부가적인 아미노산 잔기 리신 및 글리신(Lys-Gly) 또는 류신 및 글루탐산(Leu-Glu)에 의해 융합 단백질에 연결된 N-말단에서 His6-태그를 포함한다. 또다른 바람직한 구현예에서 융합 단백질은 부가적인 아미노산 잔기 리신 및 글리신(Lys-Gly) 또는 류신 및 글루탐산(Leu-Glu)에 의해 융합 단백질에 연결된 N- 및 C-말단에서 His6-태그를 포함한다.
더욱 바람직한 구현예에서 융합 단백질은 부가적인 아미노산 잔기 류신 및 글루탐산(Leu-Glu)에 의해 융합 단백질에 연결된 C-말단에서 His6-태그를 포함하고, 본 발명에 따른 융합 단백질의 펩티드 신장은 부가적인 아미노산 잔기 글리신 및 세린에 의해 효소의 N-말단에 연결된다. 또다른 바람직한 구현예에서 융합 단백질은 부가적인 아미노산 잔기 류신 및 글루탐산(Leu-Glu)에 의해 융합 단백질에 연결된 C-말단에서 His6-태그를 포함하고, 본 발명에 따른 융합 단백질의 펩티드 신장은 부가적인 아미노산 잔기 글리신 및 세린(Gly-Ser)에 의해 효소의 N-말단에 연결되고, 융합 단백질은 N-말단 상에 부가적인 아미노산 잔기 메티오닌(Met) 또는 알라닌, 메티오닌 및 글리신(Ala-Met-Gly) 또는 알라닌, 메티오닌, 글리신 및 세린(Ala-Met-Gly-Ser)을 포함한다. 바람직하게는 융합 단백질은 SEQ ID NO:77 내지 90에 따른다.
융합 단백질은 예를 들어, Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual 에 기재된 표준 클로닝 기술을 사용하여 2 개 이상의 핵산 서열을 연결시켜 제작된다. 이러한 단백질은 예를 들어, 재조합 DNA 발현 시스템에서 생성될 수 있다. 본 발명에 따른 이러한 융합 단백질은 엔돌리신에 대한 핵산 및 각각의 펩티드 신장을 융합시켜 수득될 수 있다.
본 발명에 따른 융합 단백질은 기타 부가적인 단백질에 융합되거나 연결될 수 있다. 상기 기타 부가적인 단백질의 예는 티오레독신이다.
본 발명은 추가로 본 발명에 따른 융합 단백질을 코딩하는 단리된 핵산 분자에 관한 것이다. 본 발명은 추가로 본 발명에 따른 핵산 분자를 포함하는 벡터에 관한 것이다. 상기 벡터는 상기 본 발명에 따른 융합 단백질의 구성적 또는 유도성 발현을 위해 제공될 수 있다.
본 발명은 또한 상기 융합 단백질을 발현하는 미생물, 예컨대 유전적으로 개질된 적합한 숙주 세포로부터 상기 융합 단백질을 수득하는 방법에 관한 것이다. 상기 숙주 세포는 박테리아 또는 효모 또는 동물 세포, 예를 들어, 포유동물 세포, 특히 인간 세포와 같은 미생물일 수 있다. 본 발명의 하나의 구현예에서, 숙주 세포는 피치아 파스토리스(Pichia pastoris ) 세포이다. 숙주는 단지 생물학기술적인 이유로, 예를 들어 수율, 가용성, 비용 등으로 인해 선택될 수 있지만, 또한 의료적 관점에서, 예를 들어, 비-병원성 박테리아 또는 효모, 인간 세포로부터 선택될 수 있다.
본 발명의 또다른 양상은 본 발명에 따른 융합 단백질의 발현을 수득하기 위해 적합한 숙주 세포를 유전적으로 형질전환시키는 방법에 관한 것으로, 상기 융합 단백질을 코딩하는 유전적 물질을 숙주 세포 내로 도입함으로써 숙주 세포를 유전적으로 개질시키고, 당업자에게 잘 알려진 유전자 조작 방법에 의해 이들의 번역 및 발현을 수득한다.
추가의 양상에서 본 발명은 본 발명에 따른 융합 단백질을 포함하는 조성물, 바람직하게는 약학 조성물, 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산 분자 또는 벡터로 형질전환된 숙주에 관한 것이다.
본 발명의 바람직한 구현예에서 조성물은 부가적으로, 그람-음성 박테리아의 외부 막을 투과하는 작용제, 예컨대 금속 킬레이트, 예를 들어 EDTA, TRIS, 락트산, 락토페린, 폴리마익신, 시트르산 및/또는 예를 들어, Vaara(Agents that increase the permeability of the outer membrane. Vaara M. Microbiol. Rev. 1992 Sep; 56(3):395-441)에 기재된 바와 같은 기타 성분을 포함한다. 또한 바람직한 것은 상기 언급된 투과제의 조합을 포함하는 조성물이다. 특히 바람직한 것은 약 10 μM 내지 약 100 mM EDTA, 더욱 바람직하게는 약 50 μM 내지 약 10 mM EDTA, 더욱 바람직하게는 약 0.5 mM 내지 약 10 mM EDTA, 더욱 바람직하게는 약 0.5 mM 내지 약 2 mM EDTA, 더욱 바람직하게는 약 0.5 mM 내지 1 mM EDTA 를 포함하는 조성물이다. 그러나, 또한 약 10 μM 내지 약 0.5 mM EDTA 를 포함하는 조성물이 바람직하다. 또한 바람직한 것은 약 0.5 mM 내지 약 2 mM EDTA, 더욱 바람직하게는 약 1 mM EDTA 및 부가적으로 약 10 내지 약 100 mM TRIS 를 포함하는 조성물이다.
본 발명은 또한 의약으로서 사용하기 위한, 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 숙주에 관한 것이다. 추가의 양상에서 본 발명은 그람-음성 박테리아와 연관된 질병, 질환 또는 상태의 치료 및/또는 예방을 위한 의약의 제조에서의, 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 개질된, 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산 분자를 포함하는 벡터로 형질전환된 숙주의 용도에 관한 것이다. 특히 질병, 질환 또는 상태의 치료 및/또는 예방은 엔테로박테리아세아에(Enterobacteriaceae)(에스케리챠(Escherichia), 특히 E. 콜라이(E. coli), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 특히 K. 프네우모니아에(K. pneumoniae), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia), 예르시니아(Yersinia)), 슈도모나다세아에(Pseudomonadaceae)(슈도모나스(Pseudomonas), 특히 P. 아에루기노사(P. aeruginosa), 부르크홀데리아(Burkholderia), 스테노트로포모나스(Stenotrophomonas), 쉐와넬라(Shewanella), 스핑고모나스(Sphingomonas), 코마모나스(Comamonas)), 네이세리아(Neisseria), 모라셀라(Moraxella), 비브리오(Vibrio), 아에로모나스(Aeromonas), 브루셀라(Brucella), 프란시셀라(Francisella), 보르데텔라(Bordetella), 레지오넬라(Legionella), 바르토넬라(Bartonella), 콕시엘라(Coxiella), 헤모필러스(Haemophilus), 파스퉤렐라(Pasteurella), 만헤이미아(Mannheimia), 악티노바실러스(Actinobacillus), 가르드네렐라(Gardnerella), 스피로카에타세아에(Spirochaetaceae)(트레포네마(Treponema) 및 보렐리아(Borrelia)), 렙토스피라세아에(Leptospiraceae), 캄필로박테르(Campylobacter), 헬리코박테르(Helicobacter), 스필릴룸(Spirillum), 스트렙토바실러스(Streptobacillus), 박테로이다세아에(Bacteroidaceae)(박테로이데스(Bacteroides), 푸소박테리움(Fusobacterium), 프레보텔라(Prevotella), 포르피로모나스(Porphyromonas)), 아시네토박테르(Acinetobacter), 특히 A. 바우마니이(A. baumanii)와 같은 인간 또는 동물에 대해 병원성인 균주를 포함하는 박테리아 그룹, 과, 속 또는 종의 그람-음성 박테리아에 의해 야기될 수 있다.
본 발명은 추가로 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 숙주를 포함하는 의약에 관한 것이다.
추가의 양상에서, 본 발명은 치료 및/또는 예방을 필요로 하는 대상에서의 질병, 질환 또는 상태의 치료 방법에 관한 것으로, 상기 대상에게 유효량의 본 발명에 따른 융합 단백질 및/또는 유효량의, 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 숙주 또는 본 발명에 따른 조성물을 투여하는 것을 포함한다. 대상은 인간 또는 동물일 수 있다.
특히 상기 치료 방법은 그람-음성 박테리아, 특히 상기 열거된 그람-음성 박테리아에 의해 야기되는 피부, 연조직, 호흡기, 폐, 소화관, 눈, 귀, 이, 비인두, 입, 뼈, 질, 균혈증 및/또는 심내막염 상처의 감염의 치료 및/또는 예방에 대한 것일 수 있다.
본 발명에 따른 치료(또는 예방)방법에 사용되는 투여 경로 및 투여량은 치료되는 감염의 구체적인 질환/부위에 따라 다르다. 투여 경로는 예를 들어 경구, 국소, 비인두, 비경구, 정맥내, 직장 내 또는 임의의 다른 투여 경로일 수 있다.
본 발명에 따른 융합 단백질 및/또는 유효량의, 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 숙주 또는 본 발명에 따른 조성물을 감염 부위(또는 감염될 위기에 처한 부위)에 적용하기 위해서, 제형은 감염 부위에 도달할 때까지 프로테아제, 산화, 면역 반응 등과 같은 환경적 영향으로부터 활성 화합물을 보호하는데 사용될 수 있다. 따라서, 제형은 캡슐, 당의정, 알약, 분말, 좌제, 에멀젼, 현탁액, 젤, 로션, 크림, 연고, 주사액, 시럽, 스프레이, 흡입제 또는 임의의 다른 의료적으로 합당한 생약학적 제형일 수 있다. 바람직하게는, 생약학적 제형은 적합한 담체, 안정화제, 풍미제, 완충제 또는 기타 적합한 시약을 포함할 수 있다. 예를 들어, 국소 적용을 위해 제형은 로션, 크림, 젤, 연고 또는 플라스터일 수 있으며, 비인두 적용을 위해 제형은 코에 분무에 의해 적용되는 식염수일 수 있다. 특정 감염 부위, 예를 들어 장에서의 치료 및/또는 예방의 경우에 경구 투여를 위해, 본 발명에 따른 융합 단백질을 감염 부위에 도달할 때까지 위장관의 혹독한 소화 환경으로부터 보호하는 것이 필요할 수 있다. 따라서, 위에서의 소화의 초기 단계에서 살아남고, 추후에 본 발명에 따른 융합 단백질을 장 환경 내로 분비하는 담체로서 박테리아가 사용될 수 있다.
본 발명의 구체적인 구현예에서, 슈도모나스(Pseudomonas), 특히 슈도모나스 아에루기노사(Pseudomonas aeruginosa )에 의해 야기되는 질병, 질환 또는 상태, 특히 장 영향, 특히 유아에서의 장 영향, 뇌척수막의 감염, 예를 들어, 수막염 혈반병, 중이의 감염, 피부의 감염(괴저성 농창), 특히 화상, 요로의 감염, 비염, 세균혈증 폐렴, 특히 환자가 낭성섬유증 또는 백혈병과 같은 혈액암을 앓고 있는 경우, 또는 면역억제 요법 유래의 중성구 감소증을 앓고 있는 경우의 세균혈증 폐렴, 패혈증, 특히 장기간 정맥내 또는 요로 도관삽입, 외과 수술 절차 및 중증 화상으로 인한 패혈증, 심내막염, 특히 환자가 정맥내 약물 사용자이거나 개방 심장 수술부터의 합병증을 갖는 환자인 경우의 심내막염, 고도의 파괴성 안과 감염, 특히 오염된 안과 용액의 사용후 또는 심각한 안면 화상 후 고도의 파괴성 안과 감염, 뼈연골염, 특히 심각한 외상 또는 오염된 옷을 통한 자창의 결과로서의 뼈연골염의 치료 및/또는 예방을 위한 의약의 제조에서의 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산 분자를 포함하는 벡터로 형질전환된 숙주의 용도가 제공된다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 부르크홀데리아 슈도말레이(Burkholderia pseudomallei )에 의해 야기되며, 특히 와잇모어(Whitmore)질환, 만성 폐렴, 패혈증, 특히 환자가 외상 피부 병반을 가지고 있는 경우의 패혈증이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 살모넬라 티피뮤리움(Salmonella thyphimurium ) 및 살모넬라 엔테리티디스(Salmonella enteritidis)에 의해 야기되며, 특히 급성 위창자염 및 국부적 화농성 과정, 특히 골수염, 심내막염, 담낭염 및 특히 살모넬라 티피뮤리움(Salmonella thyphimurium )에 의해 야기되는 것, 특히 환자가 2 세 미만인 경우의 수막염이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 살모넬라 티피(Salmonella typhi)에 의해 야기되며, 특히 티푸스이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 살모넬 파라티피(Salmonell paratyphi)에 의해 야기되며, 특히 파라티푸스이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 아시네토박테르 바우만니이(Acinetobacter baumannii )에 의해 야기되며, 특히 기관지염, 폐렴, 상처 감염 및 패혈증, 특히 정맥내 도관삽입 결과로서의 것이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 에스케리챠 콜라이(Escherichia coli )에 의해 야기되며, 특히 외부 장 감염, 특히 충수염, 화농성 담낭염, 복막염, 화농성 수막염 및 요로 감염, 장내 E. 콜라이(E. coli)감염, 특히 유행성 장염, 및 이질과 유사한 감염성 질환, 패혈증, 장독소혈증, 유방염 및 이질이다.
본 발명의 또다른 구체적인 구현예에서 질병, 질환 또는 상태는 클레브시엘라 프네우모니아에(Klebsiella pneumoniae)에 의해 야기되며, 특히 폐렴, 세균혈증, 수막염 및 요로 감염이다.
바람직하게는, 본 발명에 따른 융합 단백질은, 치료되는(또는 예방되는)감염이 다중내성 박테리아 균주, 특히 하기 항생제 중 하나 이상에 대한 내성이 있는 균주에 의해 야기되는 경우 의료적 치료에 사용된다: 스트렙토마이신, 테트라사이클린, 세팔로틴, 젠타마이신, 세포탁심, 세팔로스포린, 세프타지딤 또는 이미페넴. 추가로, 본 발명에 따른 융합 단백질은 통상의 항박테리아제, 예컨대 항생제, 란티바이오틱스(lantibiotics), 박테리오신 또는 엔돌리신 등과 조합되어 투여함으로써 치료 방법에 사용될 수 있다.
본 발명은 또한 하나 이상의 구획을 포함하는 약학적 패키지에 관한 것으로, 상기 하나 이상의 구획은 하나 이상의 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 하나 이상의 숙주 또는 본 발명에 따른 조성물을 포함한다.
또다른 양상에서 본 발명은 약학 조성물의 제조 방법에 관한 것이고, 상기 방법은 하나 이상의 본 발명에 따른 융합 단백질 및/또는 본 발명에 따른 융합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 핵산으로 형질전환된 하나 이상의 숙주를 약학적으로 허용가능한 희석제, 부형제 또는 담체와 혼합하는 것을 포함한다.
더욱 추가의 양상에서, 본 발명에 따른 조성물은 화장용 조성물이다. 여러 박테리아 종은 피부와 같은 환자의 신체의 환경에 노출된 표면 상에 자극을 야기할 수 있다. 이러한 자극을 방지하기 위해 또는 상기 박테리아 병원체의 미미한 소견을 제거하기 위해, 특별한 화장용 제제가 사용될 수 있고, 이것은 이미 존재하거나 새롭게 고정된 병원체 그람-음성 박테리아를 분해하기 위해 충분한 양의 본 발명에 따른 융합 단백질을 포함한다.
추가의 양상에서, 본 발명은 의료적, 식품 또는 사료 또는 환경적 진단에서의 진단 수단으로서, 특히 그람-음성 박테리아에 의해 야기된 박테리아 감염의 진단을 위한 진단 수단으로서의 용도를 위한 본 발명에 따른 융합 단백질에 관한 것이다. 이와 관련하여 본 발명에 따른 융합 단백질은 병원체 박테리아, 특히 그람-음성 병원체 박테리아를 특이적으로 분해하는 도구로서 사용될 수 있다. 본 발명에 따른 융합 단백질에 의한 박테리아 세포의 분해는 폴리마이신 B 와 같은 박테리아 세포 외피를 약화시키는 Triton X-100 또는 기타 첨가제와 같은 세제의 첨가에 의해 지원될 수 있다. 특이적 세포 분해는 PCR, 핵산 혼성화 또는 NASBA(핵산 Sequence Based Amplification)와 같은 핵산 기반 방법, IMS, 면역형광 또는 ELISA 기술과 같은 면역학적 방법, 또는 구별되는 박테리아 그룹 또는 종에 특이적인 단백질(예를 들어, 엔테로박테리아의 경우 β-갈락토시다아제, 코아귤라아제 양성 균주의 경우 코아귤라아제)을 사용하는 효소 어세이와 같은 박테리아 세포의 세포 함량에 의존하는 다른 방법을 사용하여 박테리아의 후속적인 특이적 검출을 위한 초기 단계로서 필요하다.
추가의 양상에서 본 발명은 식료품의, 식품 가공 장비의, 식품 가공 공장의, 식료품과 접촉하게 되는 표면의(예컨대 선반 및 식품 저장 장소 및 병원체, 조건 병원체 또는 기타 바람직하지 않은 박테리아가 식품 물질을 잠재적으로 감염시킬 수 있는 모든 다른 상황에서), 의료 장치의, 및 병원 및 수술 내 모든 종류의 표면의 그람-음성 박테리아 오염의 치료, 제거, 감소 또는 예방을 위한 본 발명에 따른 융합 단백질의 용도에 관한 것이다.
특히, 본 발명의 융합 단백질은 위생처리제로서 예방적으로 사용될 수 있다. 상기 위생처리제는 수술 전 후, 또는 예를 들어 혈액투석 동안에 사용될 수 있다. 게다가, 미숙 신생아 및 면역력이 약화된 사람, 또는 예방적 장치에 대한 필요성을 갖는 대상은 본 발명에 따른 융합 단백질로 치료될 수 있다. 상기 치료는 예방적으로 또는 급성 감염 동안에 이루어질 수 있다. 동일한 맥락에서, 병원 내 감염, 특히 슈도모나스 아에루기노사(Pseudomonas aeruginosa)(FQRP), 아시네토박테르(Acinetobacter)종 및 엔테로박테리아세아에(Enterobacteriaceae), 예컨대 E. 콜라이(E. coli), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia ) 및 예르시니아(Yersinia)종과 같은 항생제 내성 균주가 본 발명의 융합 단백질로 예방적으로 또는 급성기 동안 치료될 수 있을 것이다. 따라서, 본 발명에 따른 융합 단백질은 또한 세제, 텐시드, 용매, 항생제, 란티바이오틱스, 또는 박테리오신과 같은 소독 용액에 유용한 기타 성분과 조합으로 소독제로서 사용될 수 있다.
예를 들어, 병원, 치과 수술, 수의학, 부엌 또는 화장실에서 소독제로서의 본 발명에 따른 융합 단백질의 용도를 위해, 융합 단백질은 예를 들어, 유체, 분말, 젤 또는 물티슈 또는 소독 시트 제품의 성분의 형태의 조성물로 제조될 수 있다. 상기 조성물은 부가적으로, 각각의 용도 및 형태 각각에 대해 적합한 담체, 첨가제, 희석제 및/또는 부형제, 또한 EDTA 와 같이 항균 활성을 지지하는 작용제 또는 융합 단백질의 항균 활성을 향상시키는 작용제를 포함할 수 있다. 융합 단백질은 또한 통상의 소독제, 예컨대 알코올, 알데히드, 산화제, 페놀, 4 차 암모늄 화합물 또는 UV-광과 함께 사용될 수 있다. 예를 들어 표면, 대상 및/또는 장치의 소독을 위해 융합 단백질은 상기 표면, 대상 및/또는 장치 상에 적용될 수 있다. 적용은 예를 들어, 천 또는 헝겊과 같은 임의의 수단으로 소독 조성물을 적심으로써, 분사, 따름으로써 일어날 수 있다. 융합 단백질은 완전한 항균 활성을 수득하기 위해 의도되는 "반응 시간"및 각각의 적용에 따라 농도를 달리하여 사용될 수 있다.
본 발명의 또다른 양상은 본 발명이 도구 상자와 같이 사용될 수 있다는 것이다, 즉 상기 기재된 임의의 펩티드 신장은 본원에 기재된 임의의 엔돌리신, 오토리신 또는 박테리오신에 융합될 수 있다. 따라서, 각각의 박테리아에 대한 융합 단백질의 결합을 가능하게 하는 각각의 펩티드 신장, 및 각각의 박테리아의 성장을 억제하는 엔돌리신, 오토리신 또는 박테리오신을 조합하는 것이 가능하다. 따라서, 제거되어야만 하는 임의의 박테리아에 대한 적합한 융합 단백질을 제작하는 것이 가능하다.
본 발명의 이용가능성의 추가 범주는 이하 제시된 상세한 설명으로부터 명백할 것이나, 발명의 취지 및 범주 내에 있는 다양한 변형 및 개질이 본 상세한 설명으로부터 당업자에게 명백할 것이므로, 본 발명의 바람직한 구현예를 나타내면서 상세한 설명 및 특정 구현예가 오직 설명에 의해서만 제시되는 것으로 이해되어야만 할 것이다. 상기 일반적인 설명 및 하기 상세한 설명 모두는 오직 예시적이고 설명적인 것으로서 본 발명을 청구된 바와 같이 제한하고자 하는 것이 아닌 것으로 이해된다.
하기 실시예는 본 발명을 설명하나 제한하는 것으로 고려되는 것은 아니다. 다르게 지시되지 않는다면, 분자 생물학적 표준 방법, 예를 들어 [Sambrock et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York] 에 기재된 바와 같은 방법이 사용되었다.
실시예
1.
양친매성
펩티드로
개질된
gp144
및
gp188의
클로닝
, 발현 및 정제.
원리의 증명으로서, 외부 막을 통해 gp144 및 gp188을 인도하는 양친매성 펩티드의 LPS 파괴 활성의 잠재력 및 그람-음성 박테리아에 대항하는 그에 따른 항박테리아 활성이 입증된다. Gp144 및 gp188은 N-말단 펩티도글리칸 결합 및 C-말단 촉매성 도메인을 갖는 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 파지 ΦKZ 및 EL 로부터 기원하는 모듈 엔돌리신이다(Briers et al., 2007).
T4 라이소자임의 양친매성 α4 나선(SEQ ID NO: 92에 따른, aa 143-155: Pro-Asn-Arg-Ala-Lys-Arg-Val-Ile-Thr-Thr-Phe-Arg-Thr)을 코딩하는 유전자 절편을 갖는 gp144 또는 gp188 을 코딩하는 오픈 리딩 프레임의 5' 말단을 확장시키기 위해, 확장된 5' 프라이머 및 표준 3' 프라이머로의 꼬리 PCR 을 적용하였다. PCR 생성물을 pEXP5CT/TOPO® 발현 벡터(Invitrogen, Carlsbad, CA, USA)에서 클로닝하였다.
모든 제작물의 발현을 E. 콜라이(E. coli)BL21(DE3)pLysS 세포에서 수행하였다. 모든 단백질을 C-말단 6xHis-태그를 사용하는 Ni2 + 친화성 크로마토그래피에 의해 정제하였다. 상이한 정제에 대한 수율을 표 4에 제시한다. 현저하게, KZ144 와는 반대로 α4-KZ144 생성이 숙주에 대해 독성이 아니어서, 유의하게 높은 수율을 야기하였다.
정제된 저장액은 ~90% 순수하였다. 모든 gp144 유도체는 다량체 형성을 보였고, 이것은 ß-메르캅토-에탄올의 첨가에 의해 단량체로 전환될 수 있었고, 상호디술피드 결합이 다량체화를 야기하는 것을 나타낸다.
*표 4: 양친매성 펩티드로 개질된 엔돌리신의 재조합 정제의 수율*
* E. 콜라이(E. coli)발현 배양물 1 리터 당 정제된 재조합 단백질의 총 수율이 제시된다. 이 값은 단백질 농도의 분광측정계 측정 및 정제된 저장액의 총 부피에 의해 측정하였다. gp188 유도체의 정제를 고순도를 확보하기 위해 gp144 유도체(50 mM 이미다졸)와 비교하여 더욱 엄격한 조건(65 mM 이미다졸)하에서 수행하였다.
*양친매성 펩티드로 개질된 gp144 및 gp188 의 특징분석
1.A. 양친매성 펩티드로 개질된 gp144 및 gp188 의 효소적 활성
gp144 또는 gp188 의 효소적 활성에 대한 개질의 영향을 평가하기 위해, 변이체의 특이적 활성을 클로로포름-투과성이 된 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 세포 상에서 측정하고 상응하는 비개질된 엔돌리신과 비교하였다. 모든 개질된 엔돌리신의 차등 증가량을 시험하여, 상응하는 포화 곡선을 결정하였다. 상기 곡선의 선형 영역의 선형 회귀의 기울기가 특이적 활성에 대한 측정치이고, 미개질된 gp144 또는 gp188 의 기울기에 비례하여 표현되었다(표 5).
표 5: 양친매성 펩티드로 개질된 gp144 또는 gp188 의 효소적 활성*
* 상이한 변이체의 특이적 효소 활성을 측정하고, 동시에 시험된 상응하는 본래의 엔돌리신의 특이적 활성(=100%)에 비례하여 표현하였다. 어세이의 완충액 조건은 상응하는 엔돌리신의 최적 조건이었다(gp144 및 gp188 각각에 대해, KH2P04/K2HP04 I = 120 mM pH 6.2 및 I = 80 mM pH 7.3).
1.B. 양친매성 펩티드로 개질된 gp144 및 gp188 의 항박테리아 활성
기하급수상(~106/ml)의 P. 아에루기노사(P. aeruginosa)PAO1 세포를 실온에서 미개질 및 개질 gp144/gp188 과 함께 인큐베이션하였다. 1 시간 후, 세포 현탁액을 희석하고 플레이팅하였다. 밤샘 인큐베이션 후에 잔류 콜로니를 계수하였다(표 6). 미개질된 gp144 gp188 은 음성 대조군에 비해 세포 수를 유의하게 감소시키지 않았다. 상기 관찰은 장벽으로서의 외부 막의 효율을 예증한다. 양친매성 α4-나선을 갖는 융합 단백질이 α4-KZ144 및 α4-EL188 각각에 대해 50 ± 11 및 34 ± 11 %로 기하급수상 세포와 상호작용한다. 100 배 높은 밀도의 정지상 세포가 사용되었을 때, 상기 값은 유사하다(35 ± 18 및 32 ± 17%, 각각). 상이한 반복검층체 사이의 다소 높은 가변성에도 불구하고, 상기 값은 미처리된 세포와 유의하게 상이하다(α = 0.05). 일반적으로, 개질된 gp144 유도체는 gp188 유도체보다 높은 항박테리아 활성을 갖는 경향이 있다.
표 6: 엔돌리신 gp144 및 gp188 및 이들의 유도체의 항박테리아 효과*
* 기하급수적으로 성장하는 P. 아에루기노사(P. aeruginosa)PAO1 세포를 100 x 희석하고, 10 ㎍ 비투석된 단백질(최종 농도 100 ㎍/ml, 완충액: 20 mM NaH2P04-NaOH pH7.4; 0.5 M NaCl; 0.5 M 이미다졸)과 함께, 1 시간 동안 실온에서 인큐베이션하였다(최종 밀도는 ~106/ml 이었다). 분취액을 희석하고 플레이팅한다. 항박테리아 활성을 상대적 비활성화(%)(=100-(Ni/N0)*100, 식 중 N0 = 미처리된 세포의 수 및 Ni = 처리된 세포의 수임)로서 및 로그 단위(=log10N0/Ni )로 나타낸다. 모든 샘플을 6 배 반복검증하였다. 평균/표준편차를 제시한다. 통계적 분석은 스튜던트 t-검정을 사용하여 수행하였다.
실시예
2. 소수성 펩티드로
개질된
gp144
및
gp188
의
클로닝
, 발현 및 정제.
원리의 증명으로서, 외부 막을 통해 gp144 및 gp188 을 인도하는 소수성 펜타펩티드의 LPS 파괴 활성의 잠재력 및 그람-음성 박테리아에 대항하는 그에 따른 항박테리아 활성이 입증된다. Gp144 및 gp188 은 N-말단 펩티도글리칸 결합 및 C-말단 촉매성 도메인을 갖는 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 파지 ΦKZ 및 EL 로부터 기원하는 모듈 엔돌리신이다(Briers et al., 2007).
5 개의 소수성 잔기(Phe-Phe-Val-Ala-Pro)를 코딩하는 유전자 절편을 갖는 gp144 또는 gp188 을 코딩하는 오픈 리딩 프레임의 5' 말단을 확장시키기 위해, 확장된 5' 프라이머 및 표준 3' 프라이머로의 꼬리 PCR 을 적용하였다. PCR 생성물을 pEXP5CT/TOPO® 발현 벡터(Invitrogen, Carlsbad, CA, USA)에서 클로닝하였다.
모든 제작물의 발현을 E. 콜라이(E. coli)BL21(DE3)pLysS 세포에서 수행하였다. 모든 단백질을 C-말단 6xHis-태그를 사용하는 Ni2 + 친화성 크로마토그래피에 의해 정제하였다. 상이한 정제에 대한 수율을 표 7 에 제시한다.
정제된 저장액은 ~90% 순수하였다. 모든 gp144 유도체는 다량체 형성을 보였고, 이것은 ß-메르캅토-에탄올의 첨가에 의해 단량체로 전환될 수 있었고, 상호디술피드 결합이 다량체화를 야기하는 것을 나타낸다.
표 7: 엔돌리신 유도체의 재조합 정제의 수율*
* E. 콜라이(E. coli)발현 배양물 1 리터 당 정제된 재조합 단백질의 총 수율이 제시된다. 이 값은 단백질 농도의 분광측정계 측정 및 정제된 저장액의 총 부피에 의해 측정하였다. gp188 유도체의 정제를 고순도를 확보하기 위해 gp144 유도체(50 mM 이미다졸)와 비교하여 더욱 엄격한 조건(65 mM 이미다졸)하에서 수행하였다.
소수성 펜타펩티드로 개질된 gp144 및 gp188 의 특징분석
2.A. 소수성 펜타펩티드로 개질된 gp144 및 gp188 의 효소적 활성
gp144 또는 gp188 의 효소적 활성에 대한 개질의 영향을 평가하기 위해, 변이체의 특이적 활성을 클로로포름-투과성이 된 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 세포 상에서 측정하고 상응하는 비개질된 엔돌리신과 비교하였다. 모든 개질된 엔돌리신의 차등 증가량을 시험하여, 상응하는 포화 곡선을 결정하였다. 상기 곡선의 선형 영역의 선형 회귀의 기울기가 특이적 활성에 대한 측정치이고, 미개질된 gp144 또는 gp188 의 기울기에 비례하여 표현되었다(표 8).
표 8: 소수성 펩티드로 개질된 gp144 또는 gp188 의 효소적 활성*
* 상이한 변이체의 특이적 효소 활성을 측정하고, 동시에 시험된 상응하는 본래의 엔돌리신의 특이적 활성(=100%)에 비례하여 표현하였다. 어세이의 완충액 조건은 상응하는 엔돌리신의 최적 조건이었다(gp144 및 gp188 각각에 대해, KH2P04/K2HP04 I = 120 mM pH 6.2 및 I = 80 mM pH 7.3).
2.B. 소수성 펜타펩티드로 개질된 gp144 및 gp188 의 항박테리아 활성
기하급수상(~106/ml)의 P. 아에루기노사(P. aeruginosa)PAO1 세포를 실온에서 미개질 및 개질 gp144/gp188 과 함께 인큐베이션하였다. 1 시간 후, 세포 현탁액을 희석하고 플레이팅하였다. 밤샘 인큐베이션 후에 잔류 콜로니를 계수하였다(표 9). 미개질된 gp144 gp188 은 음성 대조군에 비해 세포 수를 유의하게 감소시키지 않았다. 상기 관찰은 장벽으로서의 외부 막의 효율을 예증한다. 소수성 펜타펩티드 융합 단백질로의 인큐베이션은 박테리아 세포 수(개질된 gp144 및 gp188, 각각에 대해 83 ± 7 및 69 ± 21%)의 유의한 감소(α = 0.05)를 일으킨다. 일반적으로, 개질된 gp144 유도체는 gp188 유도체보다 높은 항박테리아 활성을 갖는 경향이 있다.
표 9: 엔돌리신 gp144 및 gp188 및 이들의 유도체의 항박테리아 효과*
* 기하급수적으로 성장하는 P. 아에루기노사(P. aeruginosa)PAO1 세포를 100 x 희석하고, 10 ㎍ 비투석된 단백질(최종 농도 100 ㎍/ml, 완충액: 20 mM NaH2P04-NaOH pH7.4; 0.5 M NaCl; 0.5 M 이미다졸)과 함께, 1 시간 동안 실온에서 인큐베이션하였다(최종 밀도는 ~106/ml 이었다). 분취액을 희석하고 플레이팅한다. 항박테리아 활성을 상대적 비활성화(%)(=100-(Ni/N0)*100, 식 중 N0 = 미처리된 세포의 수 및 Ni = 처리된 세포의 수임)로서 및 로그 단위(=log10N0/Ni )로 나타낸다. 모든 샘플을 6 배 반복검증하였다. 평균/표준편차를 제시한다. 통계적 분석은 스튜던트 t-검정을 사용하여 수행하였다.
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3:
엔돌리신의
N-말단 상에서 다양한 펩티드 신장으로
개질된
KZ144 및 STM0016 의 클로닝, 발현 및 정제.
SEQ ID NO: 25에 따른 KZ144 는 N-말단 펩티도글리칸 결합 및 C-말단 촉매성 도메인을 갖는 슈도모나스 아에루기노사(Pseudomonas aeruginosa ) 파지 ΦKZ 로부터 기원하는 모듈 엔돌리신이다(Briers et al., 2007). 엔돌리신 KZ144 는 SEQ ID NO: 64에 따른 핵산 분자에 의해 코딩된다. SEQ ID NO: 64에 따른 핵산 분자는 핵산 분자의 5'-말단에서 BamH I(5'-GGA TCC-3')제한 부위 및 핵산 분자의 3'-말단에서 Xho I(5'-CTC GAG-3')제한 부위로 합성 제조되었다.
STM0016 은 E. 콜라이(E. coli) 파지 N4 엔돌리신 N4-gp61 에 대해 상동인 가상적 단백질이다. 엔돌리신 STM0016 은 SEQ ID NO: 65에 따른 핵산 분자에 의해 코딩된다. SEQ ID NO: 65에 따른 핵산 분자는 핵산 분자의 5'-말단에서 BamH I(5'-GGA TCC-3')제한 부위 및 핵산 분자의 3'-말단에서 Xho I(5'-CTC GAG-3')제한 부위로 합성 제조되었다.
N4-gp61 은 E. 콜라이(E. coli)N4 파지 엔돌리신이다. 엔돌리신은 SEQ ID NO: 91에 따른 핵산에 의해 코딩된다. SEQ ID NO: 91에 따른 핵산 분자는 핵산 분자의 5'-말단에서 BamH I(5'-GGA TCC-3')제한 부위 및 핵산 분자의 3'-말단에서 Xho I(5'-CTC GAG-3')제한 부위로 합성 제조되었다.
표 10 의 하기 펩티드 신장을 엔돌리신 KZ144 또는 STM0016 과의 융합 단백질의 제조에 사용하였다:
각각의 펩티드 신장을 코딩하는 핵산 분자는, 핵산 분자의 5'-말단에서 Nco I 제한 부위 + 2 개의 부가적인 뉴클레오티드(5'-CCA TGG GC-3')로 생성된, 스시 1 펩티드를 코딩하는 핵산 분자를 제외하고는, 핵산 분자의 5'-말단에서 Nde I(5'-CAT ATG-3')제한 부위 및 핵산 분자의 3'-말단에서 BamH I(5'-GGA TCC-3')제한 부위로 합성 제조되었다.
융합 단백질은 예를 들어, [Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual] 에 기재된 바와 같은 표준 클로닝 기술을 사용하여 2 개 이상의 핵산 서열을 연결시켜 제작된다. 따라서, 펩티드 신장을 코딩하는 핵산 분자는 각각의 제한 효소 Nde I 및 BamH I 으로의 소화물 내에서 분할되었고, 펩티드 신장 스시 1 을 코딩하는 핵산 분자의 경우에는 소화는 제한 효소 Nco I 및 BamH I 로 수행하였다. 이어서, 펩티드 신장을 코딩하는 분할된 핵산을, 또한 각각의 제한 효소 Nde I 및 BamH I 으로의 소화물 내에서 미리 분할되었던 pET21 b 발현 벡터(Novagen, Darmstadt, Germany)내로 라이게이션하였다. 펩티드 신장 스시 1 을 코딩하는 분할된 핵산 분자를, 또한 각각의 제한 효소 Nco I 및 BamH I 으로의 소화물 내에서 미리 분할되었던 개질된 pET32b 발현 벡터(비개질된 벡터는 Novagen, Darmstadt, Germany 에서 이용가능)내로 라이게이션하였다. pET32b 발현 벡터의 개질은 S-태그 및 중앙 His-태그를 코딩하는 서열의 결실을 말한다.
결국, 엔돌리신 KZ144 를 코딩하는 핵산 분자를 제한 효소 BamH I 및 Xho I 으로의 소화물 내에서 분할시켜, 엔돌리신을 또한 각각의 제한 효소 BamH I 및 Xho I 으로의 소화물 내에서 미리 분할되었던 pET21b 발현 벡터(Novagen, Darmstadt, Germany) 및 개질된 pET32b 발현 벡터 각각 내에 라이게이션할 수 있었다. 엔돌리신 STM0016 을 코딩하는 핵산 분자 및 엔돌리신 N4gp61 을 코딩하는 핵산 분자를 제한 효소 BamH I 및 Xho I 으로의 소화물 내에서 분할시켜, 각각의 엔돌리신을 pET21b 발현 벡터(Novagen, Darmstadt, Germany)내에 라이게이션할 수 있었다.
따라서, 펩티드 신장을 코딩하는 핵산 분자는 엔돌리신 KZ144 또는 STM0016 을 코딩하는 핵산 분자의 5'-말단에서 각각의 벡터 내에 라이게이션된다. 게다가, 엔돌리신 KZ144 또는 STM0016 을 코딩하는 핵산 분자는 각각의 플라스미드 내에 라이게이션되어, 6 개의 히스티딘 잔기로 이루어지는 His-태그를 코딩하는 핵산 분자가 엔돌리신을 코딩하는 핵산 분자의 3'-말단에 연합되게 된다.
일부 융합 단백질은 박테리아에서 발현시 독성이 있거나, 단백질 분해로 인해 균질하지 않을 수 있으므로, 전략은 다른 부가적인 단백질에 융합되거나 연결된 상기 융합 단백질을 발현하는 것일 수 있다. 상기 다른 부가적인 단백질에 대한 예는 티오레독신이고, 이것은 E. 콜라이(E. coli)에서 독성 항균성 펩티드의 발현을 매개하는 것으로 나타났다(TrxA mediating fusion expression of antimicrobial peptides CM4 from multiple joined genes in Escherichia coli. Zhou L, Zhao Z, Li B, Cai Y, Zhang S. Protein Expr Purif. 2009 Apr;64(2):225-230). N-말단 스시 1 펩티드 및 엔돌리신 KZ144로 이루어진 융합 단백질의 경우에는, 스시 1 펩티드는 개질된 pET32b 발현 벡터 내로 라이게이션되어, 부가적인 티오레독신이 스시 1 펩티드의 5'-말단에 연합되게 된다. 티오레독신은 엔테로키나아제의 사용에 의해 발현된 융합 단백질로부터 제거될 수 있을 것이므로, 스시 펩티드를 코딩하는 핵산 분자와 티오레독신을 코딩하는 핵산 분자 사이에 엔테로키나아제 제한 부위가 도입된다.
엔돌리신-펩티드-융합의 서열은 DNA-서열분석을 통해 통제되며, 단백질 발현을 위해 올바른 클론을 E. 콜라이(E. coli)BL21(DE3)(Novagen, Darmstadt, Germany)내로 형질전환시켰다.
SEQ ID NO:77 내지 90에 따른 융합 단백질의 재조합 발현은 E. 콜라이(E. coli)BL21(DE3)pLysS 및 E. 콜라이(E. coli)BL21(DE3) 세포(Novagen, Darmstadt, Germany)에서 수행된다. OD600nm 광학 밀도가 0.5-0.8 에 도달할 때까지 세포를 성장시켰다. 그 다음, 융합 단백질의 발현을 1 mM IPTG(이소프로필티오갈락토시드)로 유도하고, 37℃ 에서 4 시간의 기간 동안 발현을 수행하였다.
E. 콜라이(E. coli)BL21 세포를 6000g 으로 20 분 동안 원심분리에 의해 수확하고, 빙상에서 초음파분쇄를 통해 파쇄하였다. E. 콜라이(E. coli)미정제 추출물의 가용성 및 불용성 분획을 원심분리(Sorvall, SS34, 30 분, 15 000 rpm)에 의해 분리하였다. 모든 단백질을 pET21b 또는 pET32b 벡터에 의해 코딩된, C-말단 6xHis-태그를 사용하는 Ni2 + 친화성 크로마토그래피(Akta FPLC, GE Healthcare)에 의해 정제하였다.
상기 기재된 바와 같이, 융합 단백질의 일부는 관심의 단백질의 N-말단 상에 티오레독신이 융합된, 개질된 pET32b 벡터(S-태그 및 중앙 His-태그가 결실됨)를 사용하여 발현되었다. 벡터는 또한 관심의 단백질 전 우측에 엔테로키나아제 분할 부위를 함유한다. 상기 부위는 티오레독신과 관심의 단백질 사이에서 단백질 가수분해를 허용하며, 이것은 남아있는 C-말단 His-태그를 통해 정제될 수 있다. 융합 단백질 스시 1-KZ144 의 항균성 기능을 위해, 단백질 가수분해 분할에 의해 티오레독신을 제거할 필요가 있을 수 있다. 따라서, 융합 단백질은 제조자에 의해 제공되는 프로토콜에 따라 2-4 유닛/mg 재조합 엔테로키나아제(Novagen, Darmstadt, Germany)로 분할되어, 티오레독신을 제거한다. 엔테로키나아제 분할 후, 융합 단백질은 하기 기재되는 바와 같이 His-태그 정제를 통해 정제되었다.
Ni2 + 친화성 크로마토그래피를 하기와 같이 4 연속 단계로, 모두 실온에서 수행한다:
1. 3-5 ml/분의 유속으로 10 컬럼 부피 이하의 세정 완충액(20 mM 이미다졸, 1 M NaCl 및 20 mM Hepes, pH 7.4)으로 Histrap FF 5 ml 컬럼(GE Healthcare)을 평형시키는 단계.
2. 3-5 ml/분의 유속으로 Histrap FF 5 ml 컬럼 상에 총 용해물(원하는 융합 단백질을 포함하고 있음)을 로딩하는 단계.
3. 10 컬럼 부피 이하의 세정 완충액으로 컬럼을 세정하여 미결합된 샘플을 제거한 후, 3-5 ml/분의 유속으로 10% 용리 완충액(500 mM 이미다졸, 0.5 M NaCl 및 20 mM Hepes, pH 7.4)으로 세정하는 제2 세정 단계.
4. 컬럼으로부터 결합된 융합 단백질을 3-5 ml/분의 유속으로 선형 구배의, 4 컬럼 부피의 용리 완충액(500 mM 이미다졸, 0.5 M NaCl 및 20 mM Hepes, pH 7.4)으로 100% 까지 용리하는 단계.
용리 완충액(20 mM Hepes pH 7.4; 0.5 M NaCl; 500 mM 이미다졸)중의 융합 단백질의 정제된 저장액은 SDS-PAGE 젤 상에 시각적으로 측정된 바와 같이(데이터는 제시되지 않음)90% 이상 순수하였다.
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4: N-말단 상에서 다양한 펩티드 신장으로
개질된
엔돌리신
KZ144 의
항균 활성.
KZ144 및 펩티드 신장 α4 나선으로 이루어진 융합 단백질을 실시예 1 에 기재된 바와 같이 제작하였다. KZ144 및 각각의 펩티드 신장으로 이루어진 기타 융합 단백질을 실시예 3 에 기재된 바와 같이 제작하였다.
E. 콜라이(E. coli)DSMZ 11753, 아시네토박테르 바우만니이(Acinetobacter baumannii)DSMZ 30007 및 슈도모나스 아에루기노사(Pseudomonas aeruginosa )PAO1p 세포(화상 상처 단리물, Queen Astrid Hospital, Brussels; Pirnay JP et al.(2003), J Clin Microbiol ., 41(3):1192-1202)를 시험 균주로서 사용하였다. 밤샘 배양물을 신선한 LB 배지로 10 배 희석시키고, OD600=0.6 까지 성장시켰다. 배양물을 스핀 다운시키고, 희석 완충액(10 mM HEPES, 0.5 mM EDTA; pH 7.4)으로 10 배 희석시켰다. 박테리아를 실온에서 각각의 10 ㎍ 비투석된 융합 단백질과 함께 완충액(20 mM NaH2P04-NaOH pH 7.4; 0.5 M NaCl; 0.5 M 이미다졸)중의 최종 농도 100 ㎍/ml 로 인큐베이션하였다. 1 시간 후, 세포 희석 일련물을 PBS 에서 제조하고 LB 상에 플레이팅하였다. 부가적으로는, 음성 대조군을 완충액(20 mM NaH2P04-NaOH pH 7.4; 0.5 M NaCl; 0.5 M 이미다졸)을 사용하여 플레이팅하였다. 37℃ 에서 밤새 인큐베이션한 후 잔류 콜로니를 계수하였다. 계수된 세포 수에 근거하여 로그 단위(= log10N0/Ni, N0 = 미처리된 세포의 수 및 Ni = 처리된 세포의 수)로서의 항박테리아 활성을 계산하였다(표 11). 모든 샘플을 4 배 이상 반복하였다.
상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
표 11: 그람-음성 박테리아에 대항하는 다양한 펩티드 신장으로 개질된 KZ144 의 항균 활성
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5: N-말단 상에서 다양한 펩티드 신장으로
개질된
엔돌리신
STM0016
의 항균 활성
STM0016 및 펩티드 신장 사르코톡신 IA 또는 SMAP-29로 이루어진 융합 단백질을 실시예 3 에 기재된 바와 같이 제작하였다.
E. 콜라이(E. coli)DSMZ 11753, 살모넬라 티피뮤리움(Salmonella thyphimurium)DSMZ 17058 및 슈도모나스 아에루기노사(Pseudomonas aeruginosa)PAO1p 세포(화상 상처 단리물, Queen Astrid Hospital, Brussels; Pirnay JP et al.(2003), J Clin Microbiol ., 41(3):1192-1202)를 시험 균주로서 사용하였다.엔돌리신 STM0016 및 펩티드 사르코톡신 IA 또는 SMAP-29로 이루어진 융합 단백질의 항균 활성을 4 에 기재된 바와 같이 시험하였다. 상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
실시예
6: N-말단 상에서 펩티드 신장으로
개질된
엔돌리신
N4gp61
의 항균 활성
N4gp61 및 펩티드 신장 SMAP-29로 이루어진 융합 단백질을 실시예 3 에 기재된 바와 같이 제작하였다.
E. 콜라이(E. coli)DSMZ 11753, 살모넬라 티피뮤리움(Salmonella thyphimurium)DSMZ 17058 및 슈도모나스 아에루기노사(Pseudomonas aeruginosa)PAO1p 세포(화상 상처 단리물, Queen Astrid Hospital, Brussels; Pirnay JP et al.(2003), J Clin Microbiol ., 41(3):1192-1202)를 시험 균주로서 사용하였다. 엔돌리신 N4gp61 및 펩티드 SMAP-29로 이루어진 융합 단백질의 항균 활성을 실시예 4 에 기재된 바와 같이 시험하였다. 상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
실시예
7: N-말단 상에서 펩티드 신장으로
개질된
엔돌리신
gp188
의 항균 활성
엔돌리신 gp188 및 펩티드 신장 α4 나선, SMAP-29 또는 사르코톡신 IA로 이루어진 융합 단백질을 실시예 1 에 기재된 바와 같이 제작하였다. E. 콜라이(E. coli)DSMZ 11753, 아시네토박테르 바우만니이(Acinetobacter baumannii )DSMZ 30007 및 슈도모나스 아에루기노사(Pseudomonas aeruginosa )PAO1p 세포(화상 상처 단리물, Queen Astrid Hospital, Brussels; Pirnay JP et al.(2003), J Clin Microbiol., 41(3):1192-1202)를 시험 균주로서 사용하였다. 엔돌리신 gp188 및 각각의 펩티드 신장으로 이루어진 융합 단백질의 항균 활성을 실시예 4 에 기재된 바와 같이 시험하였다. 상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
실시예
8: N-말단 상에서 펩티드 신장
SMAP
-
29 로
개질된
살모넬라(
Salmonella) 엔돌리신
의 항균 활성
SEQ ID NO: 3에 따른 아미노산 서열을 갖는 살모넬라(Salmonella) 엔돌리신 및 펩티드 신장 SMAP-29로 이루어진 융합 단백질을 실시예 3 과 유사하게 제작하였다. E. 콜라이(E. coli)DSMZ 11753 및 살모넬라 티피뮤리움(Salmonella typhimurium)DSMZ 17058 을 시험 균주로서 사용하였다. 융합 단백질의 항균 활성을 실시예 4에 기재된 바와 같이 시험하였다. 상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
실시예
9: N-말단 상에서 다양한 펩티드 신장으로
개질된
아시네토박테르
바우만니이
(
Acinetobacter baumannii) 엔돌리신
의 항균 활성
SEQ ID NO: 5에 따른 아미노산 서열을 갖는 아시네토박테르 바우만니이(Acinetobacter baumannii ) 엔돌리신 및 펩티드 신장 SMAP-29, 슈딘(Pseudin)1 및 스시 1로 이루어진 융합 단백질을 실시예 3 과 유사하게 제작하였다. 아시네토박테르 바우만니이(Acinetobacter baumannii )DSMZ 30007 및 슈도모나스 아에루기노사(Pseudomonas aeruginosa )PAO1p 세포(화상 상처 단리물, Queen Astrid Hospital, Brussels; Pirnay JP et al.(2003), J Clin Microbiol ., 41(3):1192-1202)를 시험 균주로서 사용하였다. 융합 단백질의 항균 활성을 실시예 4에 기재된 바와 같이 시험하였다. 상기 융합 단백질의 항균 활성을 하기 표에 제시한다.
임의의 태그 및 링커가 없는 표 11 내지 16 중의 융합 단백질을 또한 상기 기재된 활성 어세이로 시험하였다. 이들은 모두 사용된 박테리아 균주에 대항하는 항균 활성을 보였다(데이터는 제시하지 않음).
SEQUENCE LISTING
<110> KATHOLIEKE UNIVERSITEIT LEUVEN K.U. LEUVEN R & D
<120> ANTIMICROBIAL AGENTS
<130> IP20111878DE
<150> EP 09 163 953.4
<151> 2009-06-26
<160> 100
<170> KopatentIn 2.0
<210> 1
<211> 260
<212> PRT
<213> unknown
<220>
<223> phiKZgp144
<400> 1
Met Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu
1 5 10 15
Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly
20 25 30
Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp
35 40 45
Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu
50 55 60
Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met
65 70 75 80
Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala
85 90 95
Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala
100 105 110
Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser
115 120 125
Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile
130 135 140
Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala
145 150 155 160
Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile
165 170 175
Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr
180 185 190
Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg
195 200 205
Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro
210 215 220
Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser
225 230 235 240
Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala
245 250 255
Ala His Arg Lys
260
<210> 2
<211> 292
<212> PRT
<213> unknown
<220>
<223> ELgp188
<400> 2
Met Asn Phe Arg Thr Lys Asn Gly Tyr Arg Asp Leu Gln Ala Leu Val
1 5 10 15
Lys Glu Leu Gly Leu Tyr Thr Gly Gln Ile Asp Gly Val Trp Gly Lys
20 25 30
Gly Thr Ser Ser Ser Thr Glu Thr Leu Leu Arg Gly Tyr Ala Glu Val
35 40 45
Val Gly Lys Asn Thr Gly Gly Ile Gly Leu Pro Thr Thr Ser Asp Ala
50 55 60
Ser Gly Tyr Asn Val Ile Thr Ala Leu Gln Arg Asn Leu Ala Phe Leu
65 70 75 80
Gly Leu Tyr Ser Leu Thr Val Asp Gly Ile Trp Gly Asn Gly Thr Leu
85 90 95
Ser Gly Leu Asp Lys Ala Phe Glu Val Tyr Lys Glu Arg Tyr Arg Thr
100 105 110
Pro Thr Tyr Asp Ile Ala Trp Ser Gly Lys Val Ser Pro Ala Phe Thr
115 120 125
Ala Lys Val Lys Asp Trp Cys Gly Val His Val Pro Asn His Arg Ala
130 135 140
Pro His Trp Leu Met Ala Cys Met Ala Phe Glu Thr Gly Gln Thr Phe
145 150 155 160
Ser Pro Ser Ile Lys Asn Ala Ala Gly Ser Glu Ala Tyr Gly Leu Ile
165 170 175
Gln Phe Met Ser Pro Ala Ala Asn Asp Leu Asn Val Pro Leu Ser Val
180 185 190
Ile Arg Ser Met Asp Gln Leu Thr Gln Leu Asp Leu Val Phe Lys Tyr
195 200 205
Phe Glu Met Trp Met Lys Arg Gly Lys Arg Tyr Thr Gln Leu Glu Asp
210 215 220
Phe Tyr Leu Thr Ile Phe His Pro Ala Ser Val Gly Lys Lys Ala Asp
225 230 235 240
Glu Val Leu Phe Leu Gln Gly Ser Lys Ala Tyr Leu Gln Asn Lys Gly
245 250 255
Phe Asp Val Asp Lys Asp Gly Lys Ile Thr Leu Gly Glu Ile Ser Ser
260 265 270
Thr Leu Tyr Thr Thr Tyr Tyr Lys Gly Leu Leu Pro Glu Asn Arg His
275 280 285
Val Ile Ser Tyr
290
<210> 3
<211> 181
<212> PRT
<213> unknown
<220>
<223> Salmonella endolysin
<400> 3
Met Lys Pro Lys Asp Glu Ile Phe Asp Glu Ile Leu Gly Lys Glu Gly
1 5 10 15
Gly Tyr Val Asn His Pro Asp Asp Lys Gly Gly Pro Thr Lys Trp Gly
20 25 30
Ile Thr Glu Lys Val Ala Arg Ala His Gly Tyr Arg Gly Asp Met Arg
35 40 45
Asn Leu Thr Arg Gly Gln Ala Leu Glu Ile Leu Glu Thr Asp Tyr Trp
50 55 60
Tyr Gly Pro Arg Phe Asp Arg Val Ala Lys Ala Ser Pro Asp Val Ala
65 70 75 80
Ala Glu Leu Cys Asp Thr Gly Val Asn Met Gly Pro Ser Val Ala Ala
85 90 95
Lys Met Leu Gln Arg Trp Leu Asn Val Phe Asn Gln Gly Gly Arg Leu
100 105 110
Tyr Pro Asp Met Asp Thr Asp Gly Arg Ile Gly Pro Arg Thr Leu Asn
115 120 125
Ala Leu Arg Val Tyr Leu Glu Lys Arg Gly Lys Asp Gly Glu Arg Val
130 135 140
Leu Leu Val Ala Leu Asn Cys Thr Gln Gly Glu Arg Tyr Leu Glu Leu
145 150 155 160
Ala Glu Lys Arg Glu Ala Asp Glu Ser Phe Val Tyr Gly Trp Met Lys
165 170 175
Glu Arg Val Leu Ile
180
<210> 4
<211> 163
<212> PRT
<213> unknown
<220>
<223> Enterobacteria phage T4 endolysin
<400> 4
Met Asn Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys
1 5 10 15
Ile Tyr Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu
20 25 30
Leu Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys
35 40 45
Ala Ile Gly Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu
50 55 60
Lys Leu Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg
65 70 75 80
Asn Ala Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg
85 90 95
Cys Ala Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala
100 105 110
Gly Phe Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu
115 120 125
Ala Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn
130 135 140
Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala
145 150 155 160
Tyr Lys Asn
<210> 5
<211> 280
<212> PRT
<213> unknown
<220>
<223> Acinetobacter baumanii endolysin
<400> 5
Met Glu Tyr Asp Met Ile Leu Lys Phe Gly Ser Lys Gly Asp Ala Val
1 5 10 15
Ala Thr Leu Gln Lys Gln Leu Ala Lys Met Gly Tyr Lys Gly Val Lys
20 25 30
Asp Lys Pro Leu Ser Val Asp Gly His Phe Gly Glu Ser Thr Glu Phe
35 40 45
Ala Val Ile Gln Leu Gln Arg Lys Phe Gly Leu Val Ala Asp Gly Lys
50 55 60
Val Gly Asp Lys Thr Arg Gln Ala Leu Ala Gly Asp Ser Val Ser Lys
65 70 75 80
Phe Leu Lys Asp Glu Asp Tyr Lys Lys Ala Ala Ile Arg Leu Lys Val
85 90 95
Pro Glu Leu Val Ile Arg Val Phe Gly Ala Val Glu Gly Leu Gly Val
100 105 110
Gly Phe Leu Pro Asn Gly Lys Ala Lys Ile Leu Phe Glu Arg His Arg
115 120 125
Met Tyr Phe Tyr Leu Cys Gln Ala Leu Gly Lys Thr Phe Ala Asn Ser
130 135 140
Gln Val Lys Ile Thr Pro Asn Ile Val Asn Thr Leu Thr Gly Gly Tyr
145 150 155 160
Lys Gly Asp Ala Ala Glu Tyr Thr Arg Leu Ser Met Ala Ile Asn Ile
165 170 175
His Lys Glu Ser Ala Leu Met Ser Thr Ser Trp Gly Gln Phe Gln Ile
180 185 190
Met Gly Glu Asn Trp Lys Asp Leu Gly Tyr Ser Ser Val Gln Glu Phe
195 200 205
Val Asp Gln Gln Gln Leu Asn Glu Gly Asn Gln Leu Glu Ala Phe Ile
210 215 220
Arg Phe Ile Glu Trp Lys Pro Gly Leu Leu Glu Ala Leu Arg Lys Gln
225 230 235 240
Asp Trp Asp Thr Val Phe Thr Leu Tyr Asn Gly Lys Asn Tyr Lys Lys
245 250 255
Leu Gly Tyr Gln Ala Lys Phe Gln Lys Glu Trp Asp His Leu Glu Pro
260 265 270
Ile Tyr Arg Glu Lys Thr Ala Ala
275 280
<210> 6
<211> 25
<212> PRT
<213> unknown
<220>
<223> amphipatic peptide Pleurocidin
<400> 6
Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His Val Gly Lys His Val
1 5 10 15
Gly Lys Ala Ala Leu Thr His Tyr Leu
20 25
<210> 7
<211> 31
<212> PRT
<213> unknown
<220>
<223> amphipatic peptide Cecropin P1
<400> 7
Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys
1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg
20 25 30
<210> 8
<211> 21
<212> PRT
<213> unknown
<220>
<223> amphipatic peptide Buforin II
<400> 8
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys
20
<210> 9
<211> 23
<212> PRT
<213> unknown
<220>
<223> amphipatic peptide Magainin
<400> 9
Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Val Gly Glu Ile Met Asn Ser
20
<210> 10
<211> 37
<212> PRT
<213> unknown
<220>
<223> amphipatic peptides Cathelidicine LL-37
<400> 10
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu
1 5 10 15
Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val
20 25 30
Pro Arg Thr Glu Ser
35
<210> 11
<211> 29
<212> PRT
<213> unknown
<220>
<223> SMAP-29
<400> 11
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly
20 25
<210> 12
<211> 13
<212> PRT
<213> unknown
<220>
<223> Indolicidin
<400> 12
Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg
1 5 10
<210> 13
<211> 18
<212> PRT
<213> unknown
<220>
<223> Protegrin
<400> 13
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val
1 5 10 15
Gly Arg
<210> 14
<211> 36
<212> PRT
<213> unknown
<220>
<223> Cecropin A (A.aegypti)
<400> 14
Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys Arg
1 5 10 15
Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala Lys
20 25 30
Ala Leu Arg Lys
35
<210> 15
<211> 40
<212> PRT
<213> unknown
<220>
<223> Cecropin A (D. melanogaster)
<400> 15
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Pro Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Gly
35 40
<210> 16
<211> 39
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA
<400> 16
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg
35
<210> 17
<211> 5
<212> PRT
<213> unknown
<220>
<223> Pentapeptide
<400> 17
Phe Phe Val Ala Pro
1 5
<210> 18
<211> 152
<212> PRT
<213> unknown
<220>
<223> E. coli phage K1F
<400> 18
Met Val Ser Lys Val Gln Phe Asn Pro Arg Ser Arg Thr Asp Ala Ile
1 5 10 15
Phe Val His Cys Ser Ala Thr Lys Pro Glu Met Asp Ile Gly Val Glu
20 25 30
Thr Ile Arg Met Trp His Lys Gln Gln Ala Trp Leu Asp Val Gly Tyr
35 40 45
His Phe Ile Ile Lys Arg Asp Gly Thr Val Glu Glu Gly Arg Pro Val
50 55 60
Asn Val Val Gly Ser His Val Lys Asp Trp Asn Ser Arg Ser Val Gly
65 70 75 80
Val Cys Leu Val Gly Gly Ile Asn Ala Lys Gly Gln Phe Glu Ala Asn
85 90 95
Phe Thr Pro Ala Gln Met Asn Ser Leu Arg Asn Lys Leu Asp Asp Leu
100 105 110
Lys Val Met Tyr Pro Gln Ala Glu Ile Arg Ala His His Asp Val Ala
115 120 125
Pro Lys Ala Cys Pro Ser Phe Asp Leu Gln Arg Trp Leu Ser Thr Asn
130 135 140
Glu Leu Val Thr Ser Asp Arg Gly
145 150
<210> 19
<211> 39
<212> PRT
<213> unknown
<220>
<223> Buforin I
<400> 19
Ala Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 20
<211> 165
<212> PRT
<213> unknown
<220>
<223> PSP3 gp10 Salmonella endolysin
<400> 20
Met Pro Val Ile Asn Thr His Gln Asn Ile Ala Ala Phe Leu Asp Met
1 5 10 15
Leu Ala Tyr Ser Glu Gly Thr Ala Asn His Pro Leu Thr Lys Asn Arg
20 25 30
Gly Tyr Asp Val Ile Val Thr Gly Phe Asp Gly Ser Pro Glu Ile Phe
35 40 45
Thr Asp Tyr Ser Asp His Pro Phe Ala His Gly Arg Pro Pro Lys Val
50 55 60
Phe Asn Arg Arg Gly Glu Lys Ser Thr Ala Ser Gly Arg Tyr Gln Gln
65 70 75 80
Leu Tyr Ile Phe Trp Pro His Tyr Lys Lys Gln Leu Ala Leu Pro Asp
85 90 95
Phe Ser Pro Leu Ser Gln Asp Lys Leu Ala Ile Gln Leu Ile Arg Glu
100 105 110
Arg Gly Ala Ile Asp Asp Ile Arg Ala Gly Arg Ile Glu Arg Ala Val
115 120 125
Ser Arg Cys Arg Asn Ile Trp Ala Ser Leu Pro Gly Ala Gly Tyr Gly
130 135 140
Gln Arg Glu His Ser Leu Glu Lys Leu Val Thr Val Trp Arg Thr Ala
145 150 155 160
Gly Gly Val Met Ala
165
<210> 21
<211> 165
<212> PRT
<213> unknown
<220>
<223> E. coli phage endolysin P2gp09
<400> 21
Met Pro Val Ile Asn Thr His Gln Asn Ile Ala Ala Phe Leu Asp Met
1 5 10 15
Leu Ala Val Ser Glu Gly Thr Ala Asn His Pro Leu Thr Lys Asn Arg
20 25 30
Gly Tyr Asp Val Ile Val Thr Gly Leu Asp Gly Lys Pro Glu Ile Phe
35 40 45
Thr Asp Tyr Ser Asp His Pro Phe Ala His Gly Arg Pro Ala Lys Val
50 55 60
Phe Asn Arg Arg Gly Glu Lys Ser Thr Ala Ser Gly Arg Tyr Gln Gln
65 70 75 80
Leu Tyr Leu Phe Trp Pro His Tyr Arg Lys Gln Leu Ala Leu Pro Asp
85 90 95
Phe Ser Pro Leu Ser Gln Asp Arg Leu Ala Ile Gln Leu Ile Arg Glu
100 105 110
Arg Gly Ala Leu Asp Asp Ile Arg Ala Gly Arg Ile Glu Arg Ala Ile
115 120 125
Ser Arg Cys Arg Asn Ile Trp Ala Ser Leu Pro Gly Ala Gly Tyr Gly
130 135 140
Gln Arg Glu His Ser Leu Glu Lys Leu Val Thr Val Trp Arg Thr Ala
145 150 155 160
Gly Gly Val Pro Ala
165
<210> 22
<211> 176
<212> PRT
<213> unknown
<220>
<223> STM0016
<400> 22
Asn Pro Ile Ile Asp Gly Ile Ile Ala Leu Glu Gly Gly Tyr Val Phe
1 5 10 15
Asn Pro Lys Asp Lys Gly Gly Ala Thr His Trp Gly Ile Thr Glu Ala
20 25 30
Thr Ala Arg Ala His Gly Tyr Ala Gly Asp Met Arg Asp Leu Thr His
35 40 45
Ala Glu Ala Tyr Ala Ile Leu Glu Glu Asp Tyr Trp Ile Lys Pro Gly
50 55 60
Phe Asp Val Ile Ser Thr Leu Ser Trp Pro Val Ser Phe Glu Leu Cys
65 70 75 80
Asp Ala Ala Val Asn Ile Gly Ala Tyr His Pro Ser Ala Trp Leu Gln
85 90 95
Arg Trp Leu Asn Val Phe Asn His Glu Gly Lys Arg Tyr Pro Asp Ile
100 105 110
His Val Asp Gly Asn Ile Gly Pro Arg Thr Leu Ala Ala Leu Glu His
115 120 125
Tyr Leu Ala Trp Arg Gly Gln Glu Gly Glu Ala Val Leu Val Lys Ala
130 135 140
Leu Asn Cys Ser Gln Gly Thr Tyr Tyr Leu Asn Val Ala Glu Lys Asn
145 150 155 160
His Asn Asn Glu Gln Phe Ile Tyr Gly Trp Ile Lys Asn Arg Val Thr
165 170 175
<210> 23
<211> 208
<212> PRT
<213> unknown
<220>
<223> E.coli Phage N4 muramidase N4-gp61
<400> 23
Met Ala Ile Ser Lys Lys Lys Val Gly Gly Val Gly Gly Val Ile Ala
1 5 10 15
Ala Ile Ile Ala Ala Val Phe Ala Val Glu Gly Gly Tyr Val Asn Asp
20 25 30
Pro Lys Asp Pro Gly Gly Glu Thr Asn His Gly Val Thr Ile Gln Val
35 40 45
Ala Gln Lys His Lys Gln Glu Leu Glu Ser Met Tyr Asn Trp Asp Gly
50 55 60
Ser Met Lys Asn Leu Thr Gln Glu Met Ala Ser Ser Ile Tyr Tyr Asn
65 70 75 80
Asp Tyr Ile Leu Lys Pro Gly Phe Val Lys Phe Ala Asp Val Ser Pro
85 90 95
Ala Val Thr Glu Lys Leu Val Asp Ala Gly Val Asn Thr Gly Pro Ala
100 105 110
Arg Pro Ser Arg Trp Leu Gln Glu Ser Leu Asn Ala Phe Ser Arg Asn
115 120 125
Gly Lys Asp Tyr Pro Lys Ile Gln Val Asp Gly Lys Val Gly Ser Gly
130 135 140
Thr Leu Ser Ala Tyr Lys Ser Leu Gln Asn Lys Arg Gly Lys Val Glu
145 150 155 160
Ala Cys Lys Leu Ile Leu Lys Ser Leu Asp Gly Lys Gln Leu Asn Tyr
165 170 175
Tyr Leu Ser Leu Asn Met Pro Glu Tyr Thr Thr Gly Trp Ile Ala Asn
180 185 190
Arg Ile Gly Asn Val Pro Leu Glu Arg Cys Asn Glu Asp Ile Val Asn
195 200 205
<210> 24
<211> 184
<212> PRT
<213> unknown
<220>
<223> 24 N4-gp61 trunc.
<400> 24
Val Glu Gly Gly Tyr Val Asn Asp Pro Lys Asp Pro Gly Gly Glu Thr
1 5 10 15
Asn His Gly Val Thr Ile Gln Val Ala Gln Lys His Lys Gln Glu Leu
20 25 30
Glu Ser Met Tyr Asn Trp Asp Gly Ser Met Lys Asn Leu Thr Gln Glu
35 40 45
Met Ala Ser Ser Ile Tyr Tyr Asn Asp Tyr Ile Leu Lys Pro Gly Phe
50 55 60
Val Lys Phe Ala Asp Val Ser Pro Ala Val Thr Glu Lys Leu Val Asp
65 70 75 80
Ala Gly Val Asn Thr Gly Pro Ala Arg Pro Ser Arg Trp Leu Gln Glu
85 90 95
Ser Leu Asn Ala Phe Ser Arg Asn Gly Lys Asp Tyr Pro Lys Ile Gln
100 105 110
Val Asp Gly Lys Val Gly Ser Gly Thr Leu Ser Ala Tyr Lys Ser Leu
115 120 125
Gln Asn Lys Arg Gly Lys Val Glu Ala Cys Lys Leu Ile Leu Lys Ser
130 135 140
Leu Asp Gly Lys Gln Leu Asn Tyr Tyr Leu Ser Leu Asn Met Pro Glu
145 150 155 160
Tyr Thr Thr Gly Trp Ile Ala Asn Arg Ile Gly Asn Val Pro Leu Glu
165 170 175
Arg Cys Asn Glu Asp Ile Val Asn
180
<210> 25
<211> 259
<212> PRT
<213> unknown
<220>
<223> E.coli phage KZ144
<400> 25
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln
1 5 10 15
Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
20 25 30
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
35 40 45
Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
50 55 60
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
65 70 75 80
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
85 90 95
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
100 105 110
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
115 120 125
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
130 135 140
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
145 150 155 160
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
165 170 175
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
180 185 190
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
195 200 205
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
210 215 220
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
225 230 235 240
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
245 250 255
His Arg Lys
<210> 26
<211> 22
<212> PRT
<213> unknown
<220>
<223> Nigrocine 2
<400> 26
Gly Leu Leu Ser Lys Val Leu Gly Val Gly Lys Lys Val Leu Cys Gly
1 5 10 15
Val Ser Gly Leu Val Cys
20
<210> 27
<211> 24
<212> PRT
<213> unknown
<220>
<223> Ascaphine 5
<400> 27
Gly Ile Lys Asp Trp Ile Lys Gly Ala Ala Lys Lys Leu Ile Lys Thr
1 5 10 15
Val Ala Ser His Ile Ala Asn Gln
20
<210> 28
<211> 17
<212> PRT
<213> unknown
<220>
<223> Apidaecin
<400> 28
Ala Asn Arg Pro Val Tyr Ile Pro Pro Pro Arg Pro Pro His Pro Arg
1 5 10 15
Leu
<210> 29
<211> 24
<212> PRT
<213> unknown
<220>
<223> Pseudin 1
<400> 29
Gly Leu Asn Thr Leu Lys Lys Val Phe Gln Gly Leu His Glu Ala Ile
1 5 10 15
Lys Leu Ile Asn Asn His Val Gln
20
<210> 30
<211> 18
<212> PRT
<213> unknown
<220>
<223> Ranalexin
<400> 30
Phe Leu Gly Gly Leu Ile Val Pro Ala Met Ile Cys Ala Val Thr Lys
1 5 10 15
Lys Cys
<210> 31
<211> 26
<212> PRT
<213> unknown
<220>
<223> Melittin
<400> 31
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
<210> 32
<211> 34
<212> PRT
<213> unknown
<220>
<223> Sushi 1
<400> 32
Gly Phe Lys Leu Lys Gly Met Ala Arg Ile Ser Cys Leu Pro Asn Gly
1 5 10 15
Gln Trp Ser Asn Phe Pro Pro Lys Cys Ile Arg Glu Cys Ala Met Val
20 25 30
Ser Ser
<210> 33
<211> 27
<212> PRT
<213> unknown
<220>
<223> WLUB2 variant
<400> 33
Lys Arg Trp Val Lys Arg Val Lys Arg Val Lys Arg Trp Val Lys Arg
1 5 10 15
Val Val Arg Val Val Lys Arg Trp Val Lys Arg
20 25
<210> 34
<211> 332
<212> PRT
<213> unknown
<220>
<223> OBPgpLYS
<400> 34
Met Gly Ser Lys Asn Ser Glu Lys Asn Ala Ser Ile Ile Met Ser Ile
1 5 10 15
Gln Arg Thr Leu Ala Ser Leu Ser Leu Tyr Gly Gly Arg Ile Asp Gly
20 25 30
Leu Phe Gly Glu Lys Cys Arg Gly Ala Ile Ile Leu Met Leu Asn Lys
35 40 45
Val Tyr Pro Asn Phe Ser Thr Asn Lys Leu Pro Ser Asn Thr Tyr Glu
50 55 60
Ala Glu Ser Val Phe Thr Phe Leu Gln Thr Ala Leu Ala Gly Val Gly
65 70 75 80
Leu Tyr Thr Ile Thr Ile Asp Gly Lys Trp Gly Gly Thr Ser Gln Gly
85 90 95
Ala Ile Asp Ala Leu Val Lys Ser Tyr Arg Gln Ile Thr Glu Ala Glu
100 105 110
Arg Ala Gly Ser Thr Leu Pro Leu Gly Leu Ala Thr Val Met Ser Lys
115 120 125
His Met Ser Ile Glu Gln Leu Arg Ala Met Leu Pro Thr Asp Arg Gln
130 135 140
Gly Tyr Ala Glu Val Tyr Ile Asp Pro Leu Asn Glu Thr Met Asp Ile
145 150 155 160
Phe Glu Ile Asn Thr Pro Leu Arg Ile Ala His Phe Met Ala Gln Ile
165 170 175
Leu His Glu Thr Ala Cys Phe Lys Tyr Thr Glu Glu Leu Ala Ser Gly
180 185 190
Lys Ala Tyr Glu Gly Arg Ala Asp Leu Gly Asn Thr Arg Pro Gly Asp
195 200 205
Gly Pro Leu Phe Lys Gly Arg Gly Leu Leu Gln Ile Thr Gly Arg Leu
210 215 220
Asn Tyr Val Lys Cys Gln Val Tyr Leu Arg Glu Lys Leu Lys Asp Pro
225 230 235 240
Thr Phe Asp Ile Thr Ser Ser Val Thr Cys Ala Gln Gln Leu Ser Glu
245 250 255
Ser Pro Leu Leu Ala Ala Leu Ala Ser Gly Tyr Phe Trp Arg Phe Ile
260 265 270
Lys Pro Lys Leu Asn Glu Thr Ala Asp Lys Asp Asp Ile Tyr Trp Val
275 280 285
Ser Val Tyr Val Asn Gly Tyr Ala Lys Gln Ala Asn Pro Tyr Tyr Pro
290 295 300
Asn Arg Asp Lys Glu Pro Asn His Met Lys Glu Arg Val Gln Met Leu
305 310 315 320
Ala Val Thr Lys Lys Ala Leu Gly Ile Val Lys Gly
325 330
<210> 35
<211> 18
<212> PRT
<213> unknown
<220>
<223> 35 Walmagh1
<400> 35
Gly Phe Phe Ile Pro Ala Val Ile Leu Pro Ser Ile Ala Phe Leu Ile
1 5 10 15
Val Pro
<210> 36
<211> 283
<212> PRT
<213> unknown
<220>
<223> Ascaphine5-KZ144
<400> 36
Gly Ile Lys Asp Trp Ile Lys Gly Ala Ala Lys Lys Leu Ile Lys Thr
1 5 10 15
Val Ala Ser His Ile Ala Asn Gln Lys Val Leu Arg Lys Gly Asp Arg
20 25 30
Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr
35 40 45
Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln
50 55 60
Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val
65 70 75 80
Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile
85 90 95
Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala
100 105 110
Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser
115 120 125
Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu
130 135 140
Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr
145 150 155 160
Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val
165 170 175
Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala
180 185 190
Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro
195 200 205
Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe
210 215 220
Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu
225 230 235 240
Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile
245 250 255
Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn
260 265 270
Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 37
<211> 276
<212> PRT
<213> unknown
<220>
<223> Apidaecine-KZ144
<400> 37
Ala Asn Arg Pro Val Tyr Ile Pro Pro Pro Arg Pro Pro His Pro Arg
1 5 10 15
Leu Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu
20 25 30
Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly
35 40 45
Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp
50 55 60
Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu
65 70 75 80
Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met
85 90 95
Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala
100 105 110
Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala
115 120 125
Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser
130 135 140
Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile
145 150 155 160
Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala
165 170 175
Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile
180 185 190
Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr
195 200 205
Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg
210 215 220
Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro
225 230 235 240
Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser
245 250 255
Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala
260 265 270
Ala His Arg Lys
275
<210> 38
<211> 281
<212> PRT
<213> unknown
<220>
<223> Nigrocine2-KZ144
<400> 38
Gly Leu Leu Ser Lys Val Leu Gly Val Gly Lys Lys Val Leu Cys Gly
1 5 10 15
Val Ser Gly Leu Val Cys Lys Val Leu Arg Lys Gly Asp Arg Gly Asp
20 25 30
Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val
35 40 45
Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val
50 55 60
Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys
65 70 75 80
Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr
85 90 95
Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr
100 105 110
Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu
115 120 125
Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys
130 135 140
Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr
145 150 155 160
Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr
165 170 175
Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met
180 185 190
Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu
195 200 205
Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly
210 215 220
Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala
225 230 235 240
Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr
245 250 255
Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met
260 265 270
Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 39
<211> 283
<212> PRT
<213> unknown
<220>
<223> Pseudin1-KZ144
<400> 39
Gly Leu Asn Thr Leu Lys Lys Val Phe Gln Gly Leu His Glu Ala Ile
1 5 10 15
Lys Leu Ile Asn Asn His Val Gln Lys Val Leu Arg Lys Gly Asp Arg
20 25 30
Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr
35 40 45
Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln
50 55 60
Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val
65 70 75 80
Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile
85 90 95
Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala
100 105 110
Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser
115 120 125
Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu
130 135 140
Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr
145 150 155 160
Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val
165 170 175
Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala
180 185 190
Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro
195 200 205
Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe
210 215 220
Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu
225 230 235 240
Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile
245 250 255
Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn
260 265 270
Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 40
<211> 277
<212> PRT
<213> unknown
<220>
<223> Ranalexin-KZ144
<400> 40
Phe Leu Gly Gly Leu Ile Val Pro Ala Met Ile Cys Ala Val Thr Lys
1 5 10 15
Lys Cys Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln
20 25 30
Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp
35 40 45
Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys
50 55 60
Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala
65 70 75 80
Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro
85 90 95
Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn
100 105 110
Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe
115 120 125
Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser
130 135 140
Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met
145 150 155 160
Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly
165 170 175
Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu
180 185 190
Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro
195 200 205
Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala
210 215 220
Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe
225 230 235 240
Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly
245 250 255
Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val
260 265 270
Ala Ala His Arg Lys
275
<210> 41
<211> 286
<212> PRT
<213> unknown
<220>
<223> WLBU2-Variant-KZ144
<400> 41
Lys Arg Trp Val Lys Arg Val Lys Arg Val Lys Arg Trp Val Lys Arg
1 5 10 15
Val Val Arg Val Val Lys Arg Trp Val Lys Arg Lys Val Leu Arg Lys
20 25 30
Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu
35 40 45
Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr
50 55 60
Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp
65 70 75 80
Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser
85 90 95
Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser
100 105 110
Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly
115 120 125
Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe
130 135 140
Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln
145 150 155 160
Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys
165 170 175
Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg
180 185 190
Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile
195 200 205
Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu
210 215 220
Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly
225 230 235 240
Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn
245 250 255
Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu
260 265 270
Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
<210> 42
<211> 293
<212> PRT
<213> unknown
<220>
<223> Sushi1-KZ144
<400> 42
Gly Phe Lys Leu Lys Gly Met Ala Arg Ile Ser Cys Leu Pro Asn Gly
1 5 10 15
Gln Trp Ser Asn Phe Pro Pro Lys Cys Ile Arg Glu Cys Ala Met Val
20 25 30
Ser Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln
35 40 45
Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp
50 55 60
Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys
65 70 75 80
Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala
85 90 95
Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro
100 105 110
Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn
115 120 125
Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe
130 135 140
Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser
145 150 155 160
Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met
165 170 175
Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly
180 185 190
Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu
195 200 205
Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro
210 215 220
Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala
225 230 235 240
Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe
245 250 255
Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly
260 265 270
Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val
275 280 285
Ala Ala His Arg Lys
290
<210> 43
<211> 285
<212> PRT
<213> unknown
<220>
<223> Melttin-KZ144
<400> 43
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Lys Val Leu Arg Lys Gly
20 25 30
Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys
35 40 45
Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe
50 55 60
Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly
65 70 75 80
Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro
85 90 95
Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg
100 105 110
Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val
115 120 125
Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp
130 135 140
Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe
145 150 155 160
Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr
165 170 175
Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile
180 185 190
Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu
195 200 205
Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala
210 215 220
His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln
225 230 235 240
Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro
245 250 255
Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val
260 265 270
Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
<210> 44
<211> 296
<212> PRT
<213> unknown
<220>
<223> LL-37-KZ144
<400> 44
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu
1 5 10 15
Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val
20 25 30
Pro Arg Thr Glu Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu
35 40 45
Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly
50 55 60
Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys
65 70 75 80
Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn
85 90 95
Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys
100 105 110
Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro
115 120 125
Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu
130 135 140
Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala
145 150 155 160
Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp
165 170 175
Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp
180 185 190
Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly
195 200 205
Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys
210 215 220
Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro
225 230 235 240
Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala
245 250 255
Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn
260 265 270
Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp
275 280 285
Gly Lys Val Ala Ala His Arg Lys
290 295
<210> 45
<211> 272
<212> PRT
<213> unknown
<220>
<223> Indolicidin-KZ144
<400> 45
Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Val Leu
1 5 10 15
Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu
20 25 30
Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn
35 40 45
Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp
50 55 60
Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys
65 70 75 80
Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn
85 90 95
Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala
100 105 110
Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser
115 120 125
Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp
130 135 140
Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly
145 150 155 160
Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp
165 170 175
Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met
180 185 190
Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu
195 200 205
Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr
210 215 220
Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln
225 230 235 240
Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile
245 250 255
Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
260 265 270
<210> 46
<211> 288
<212> PRT
<213> unknown
<220>
<223> SMAP-29-KZ144
<400> 46
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Lys Val Leu
20 25 30
Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu
35 40 45
Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn
50 55 60
Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp
65 70 75 80
Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys
85 90 95
Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn
100 105 110
Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala
115 120 125
Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser
130 135 140
Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp
145 150 155 160
Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly
165 170 175
Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp
180 185 190
Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met
195 200 205
Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu
210 215 220
Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr
225 230 235 240
Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln
245 250 255
Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile
260 265 270
Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
<210> 47
<211> 277
<212> PRT
<213> unknown
<220>
<223> Protegrin-KZ144
<400> 47
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val
1 5 10 15
Gly Arg Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln
20 25 30
Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp
35 40 45
Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys
50 55 60
Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala
65 70 75 80
Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro
85 90 95
Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn
100 105 110
Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe
115 120 125
Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser
130 135 140
Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met
145 150 155 160
Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly
165 170 175
Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu
180 185 190
Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro
195 200 205
Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala
210 215 220
Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe
225 230 235 240
Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly
245 250 255
Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val
260 265 270
Ala Ala His Arg Lys
275
<210> 48
<211> 290
<212> PRT
<213> unknown
<220>
<223> Cecropin P1-KZ144
<400> 48
Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys
1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg Lys
20 25 30
Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr
35 40 45
Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe
50 55 60
Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys
65 70 75 80
Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe
85 90 95
Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr
100 105 110
Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu
115 120 125
Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile
130 135 140
Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr
145 150 155 160
Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn
165 170 175
Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg
180 185 190
Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu
195 200 205
Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr
210 215 220
Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe
225 230 235 240
Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu
245 250 255
Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys
260 265 270
Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His
275 280 285
Arg Lys
290
<210> 49
<211> 282
<212> PRT
<213> unknown
<220>
<223> Magainin-KZ144
<400> 49
Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Val Gly Glu Ile Met Asn Ser Lys Val Leu Arg Lys Gly Asp Arg Gly
20 25 30
Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp
35 40 45
Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val
50 55 60
Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly
65 70 75 80
Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro
85 90 95
Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala
100 105 110
Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln
115 120 125
Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile
130 135 140
Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly
145 150 155 160
Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu
165 170 175
Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu
180 185 190
Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val
195 200 205
Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe
210 215 220
Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu
225 230 235 240
Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe
245 250 255
Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu
260 265 270
Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 50
<211> 284
<212> PRT
<213> unknown
<220>
<223> Pleurocidin-KZ144
<400> 50
Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His Val Gly Lys His Val
1 5 10 15
Gly Lys Ala Ala Leu Thr His Tyr Leu Lys Val Leu Arg Lys Gly Asp
20 25 30
Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly
35 40 45
Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn
50 55 60
Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile
65 70 75 80
Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro
85 90 95
Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala
100 105 110
Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg
115 120 125
Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr
130 135 140
Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu
145 150 155 160
Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly
165 170 175
Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser
180 185 190
Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg
195 200 205
Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His
210 215 220
Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn
225 230 235 240
Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser
245 250 255
Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr
260 265 270
Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 51
<211> 295
<212> PRT
<213> unknown
<220>
<223> Cecropin A (A.aegypti)-KZ144
<400> 51
Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys Arg
1 5 10 15
Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala Lys
20 25 30
Ala Leu Arg Lys Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val
35 40 45
Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys
50 55 60
Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe
65 70 75 80
Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr
85 90 95
Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr
100 105 110
Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val
115 120 125
Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu
130 135 140
Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys
145 150 155 160
Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys
165 170 175
Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro
180 185 190
Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala
195 200 205
Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg
210 215 220
Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly
225 230 235 240
Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr
245 250 255
His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys
260 265 270
Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly
275 280 285
Lys Val Ala Ala His Arg Lys
290 295
<210> 52
<211> 299
<212> PRT
<213> unknown
<220>
<223> Cecropin A (D.melanogaster)-KZ144
<400> 52
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Pro Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Gly Lys Val Leu Arg Lys Gly Asp Arg
35 40 45
Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr
50 55 60
Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln
65 70 75 80
Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val
85 90 95
Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile
100 105 110
Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala
115 120 125
Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser
130 135 140
Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu
145 150 155 160
Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr
165 170 175
Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val
180 185 190
Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala
195 200 205
Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro
210 215 220
Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe
225 230 235 240
Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu
245 250 255
Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile
260 265 270
Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn
275 280 285
Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
290 295
<210> 53
<211> 280
<212> PRT
<213> unknown
<220>
<223> BuforinII-KZ144
<400> 53
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu
20 25 30
Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly
35 40 45
Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys
50 55 60
Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn
65 70 75 80
Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys
85 90 95
Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro
100 105 110
Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu
115 120 125
Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala
130 135 140
Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp
145 150 155 160
Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp
165 170 175
Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly
180 185 190
Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys
195 200 205
Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro
210 215 220
Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala
225 230 235 240
Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn
245 250 255
Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp
260 265 270
Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 54
<211> 298
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA-KZ144
<400> 54
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Lys Val Leu Arg Lys Gly Asp Arg Gly
35 40 45
Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp
50 55 60
Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val
65 70 75 80
Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly
85 90 95
Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro
100 105 110
Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala
115 120 125
Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln
130 135 140
Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile
145 150 155 160
Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly
165 170 175
Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu
180 185 190
Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu
195 200 205
Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val
210 215 220
Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe
225 230 235 240
Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu
245 250 255
Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe
260 265 270
Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu
275 280 285
Met Asp Gly Lys Val Ala Ala His Arg Lys
290 295
<210> 55
<211> 200
<212> PRT
<213> unknown
<220>
<223> Ascaphine 5-STM0016
<400> 55
Gly Ile Lys Asp Trp Ile Lys Gly Ala Ala Lys Lys Leu Ile Lys Thr
1 5 10 15
Val Ala Ser His Ile Ala Asn Gln Asn Pro Ile Ile Asp Gly Ile Ile
20 25 30
Ala Leu Glu Gly Gly Tyr Val Phe Asn Pro Lys Asp Lys Gly Gly Ala
35 40 45
Thr His Trp Gly Ile Thr Glu Ala Thr Ala Arg Ala His Gly Tyr Ala
50 55 60
Gly Asp Met Arg Asp Leu Thr His Ala Glu Ala Tyr Ala Ile Leu Glu
65 70 75 80
Glu Asp Tyr Trp Ile Lys Pro Gly Phe Asp Val Ile Ser Thr Leu Ser
85 90 95
Trp Pro Val Ser Phe Glu Leu Cys Asp Ala Ala Val Asn Ile Gly Ala
100 105 110
Tyr His Pro Ser Ala Trp Leu Gln Arg Trp Leu Asn Val Phe Asn His
115 120 125
Glu Gly Lys Arg Tyr Pro Asp Ile His Val Asp Gly Asn Ile Gly Pro
130 135 140
Arg Thr Leu Ala Ala Leu Glu His Tyr Leu Ala Trp Arg Gly Gln Glu
145 150 155 160
Gly Glu Ala Val Leu Val Lys Ala Leu Asn Cys Ser Gln Gly Thr Tyr
165 170 175
Tyr Leu Asn Val Ala Glu Lys Asn His Asn Asn Glu Gln Phe Ile Tyr
180 185 190
Gly Trp Ile Lys Asn Arg Val Thr
195 200
<210> 56
<211> 198
<212> PRT
<213> unknown
<220>
<223> Nigrocine2-STM0016
<400> 56
Gly Leu Leu Ser Lys Val Leu Gly Val Gly Lys Lys Val Leu Cys Gly
1 5 10 15
Val Ser Gly Leu Val Cys Asn Pro Ile Ile Asp Gly Ile Ile Ala Leu
20 25 30
Glu Gly Gly Tyr Val Phe Asn Pro Lys Asp Lys Gly Gly Ala Thr His
35 40 45
Trp Gly Ile Thr Glu Ala Thr Ala Arg Ala His Gly Tyr Ala Gly Asp
50 55 60
Met Arg Asp Leu Thr His Ala Glu Ala Tyr Ala Ile Leu Glu Glu Asp
65 70 75 80
Tyr Trp Ile Lys Pro Gly Phe Asp Val Ile Ser Thr Leu Ser Trp Pro
85 90 95
Val Ser Phe Glu Leu Cys Asp Ala Ala Val Asn Ile Gly Ala Tyr His
100 105 110
Pro Ser Ala Trp Leu Gln Arg Trp Leu Asn Val Phe Asn His Glu Gly
115 120 125
Lys Arg Tyr Pro Asp Ile His Val Asp Gly Asn Ile Gly Pro Arg Thr
130 135 140
Leu Ala Ala Leu Glu His Tyr Leu Ala Trp Arg Gly Gln Glu Gly Glu
145 150 155 160
Ala Val Leu Val Lys Ala Leu Asn Cys Ser Gln Gly Thr Tyr Tyr Leu
165 170 175
Asn Val Ala Glu Lys Asn His Asn Asn Glu Gln Phe Ile Tyr Gly Trp
180 185 190
Ile Lys Asn Arg Val Thr
195
<210> 57
<211> 205
<212> PRT
<213> unknown
<220>
<223> SMAP-29-STM0016
<400> 57
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Asn Pro Ile
20 25 30
Ile Asp Gly Ile Ile Ala Leu Glu Gly Gly Tyr Val Phe Asn Pro Lys
35 40 45
Asp Lys Gly Gly Ala Thr His Trp Gly Ile Thr Glu Ala Thr Ala Arg
50 55 60
Ala His Gly Tyr Ala Gly Asp Met Arg Asp Leu Thr His Ala Glu Ala
65 70 75 80
Tyr Ala Ile Leu Glu Glu Asp Tyr Trp Ile Lys Pro Gly Phe Asp Val
85 90 95
Ile Ser Thr Leu Ser Trp Pro Val Ser Phe Glu Leu Cys Asp Ala Ala
100 105 110
Val Asn Ile Gly Ala Tyr His Pro Ser Ala Trp Leu Gln Arg Trp Leu
115 120 125
Asn Val Phe Asn His Glu Gly Lys Arg Tyr Pro Asp Ile His Val Asp
130 135 140
Gly Asn Ile Gly Pro Arg Thr Leu Ala Ala Leu Glu His Tyr Leu Ala
145 150 155 160
Trp Arg Gly Gln Glu Gly Glu Ala Val Leu Val Lys Ala Leu Asn Cys
165 170 175
Ser Gln Gly Thr Tyr Tyr Leu Asn Val Ala Glu Lys Asn His Asn Asn
180 185 190
Glu Gln Phe Ile Tyr Gly Trp Ile Lys Asn Arg Val Thr
195 200 205
<210> 58
<211> 215
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA-STM0016
<400> 58
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Asn Pro Ile Ile Asp Gly Ile Ile Ala
35 40 45
Leu Glu Gly Gly Tyr Val Phe Asn Pro Lys Asp Lys Gly Gly Ala Thr
50 55 60
His Trp Gly Ile Thr Glu Ala Thr Ala Arg Ala His Gly Tyr Ala Gly
65 70 75 80
Asp Met Arg Asp Leu Thr His Ala Glu Ala Tyr Ala Ile Leu Glu Glu
85 90 95
Asp Tyr Trp Ile Lys Pro Gly Phe Asp Val Ile Ser Thr Leu Ser Trp
100 105 110
Pro Val Ser Phe Glu Leu Cys Asp Ala Ala Val Asn Ile Gly Ala Tyr
115 120 125
His Pro Ser Ala Trp Leu Gln Arg Trp Leu Asn Val Phe Asn His Glu
130 135 140
Gly Lys Arg Tyr Pro Asp Ile His Val Asp Gly Asn Ile Gly Pro Arg
145 150 155 160
Thr Leu Ala Ala Leu Glu His Tyr Leu Ala Trp Arg Gly Gln Glu Gly
165 170 175
Glu Ala Val Leu Val Lys Ala Leu Asn Cys Ser Gln Gly Thr Tyr Tyr
180 185 190
Leu Asn Val Ala Glu Lys Asn His Asn Asn Glu Gln Phe Ile Tyr Gly
195 200 205
Trp Ile Lys Asn Arg Val Thr
210 215
<210> 59
<211> 233
<212> PRT
<213> unknown
<220>
<223> Melittin-N4gp61
<400> 59
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Ala Ile Ser Lys Lys Lys
20 25 30
Val Gly Gly Val Gly Gly Val Ile Ala Ala Ile Ile Ala Ala Val Phe
35 40 45
Ala Val Glu Gly Gly Tyr Val Asn Asp Pro Lys Asp Pro Gly Gly Glu
50 55 60
Thr Asn His Gly Val Thr Ile Gln Val Ala Gln Lys His Lys Gln Glu
65 70 75 80
Leu Glu Ser Met Tyr Asn Trp Asp Gly Ser Met Lys Asn Leu Thr Gln
85 90 95
Glu Met Ala Ser Ser Ile Tyr Tyr Asn Asp Tyr Ile Leu Lys Pro Gly
100 105 110
Phe Val Lys Phe Ala Asp Val Ser Pro Ala Val Thr Glu Lys Leu Val
115 120 125
Asp Ala Gly Val Asn Thr Gly Pro Ala Arg Pro Ser Arg Trp Leu Gln
130 135 140
Glu Ser Leu Asn Ala Phe Ser Arg Asn Gly Lys Asp Tyr Pro Lys Ile
145 150 155 160
Gln Val Asp Gly Lys Val Gly Ser Gly Thr Leu Ser Ala Tyr Lys Ser
165 170 175
Leu Gln Asn Lys Arg Gly Lys Val Glu Ala Cys Lys Leu Ile Leu Lys
180 185 190
Ser Leu Asp Gly Lys Gln Leu Asn Tyr Tyr Leu Ser Leu Asn Met Pro
195 200 205
Glu Tyr Thr Thr Gly Trp Ile Ala Asn Arg Ile Gly Asn Val Pro Leu
210 215 220
Glu Arg Cys Asn Glu Asp Ile Val Asn
225 230
<210> 60
<211> 236
<212> PRT
<213> unknown
<220>
<223> SMAP-29-N4gp61
<400> 60
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Ala Ile Ser
20 25 30
Lys Lys Lys Val Gly Gly Val Gly Gly Val Ile Ala Ala Ile Ile Ala
35 40 45
Ala Val Phe Ala Val Glu Gly Gly Tyr Val Asn Asp Pro Lys Asp Pro
50 55 60
Gly Gly Glu Thr Asn His Gly Val Thr Ile Gln Val Ala Gln Lys His
65 70 75 80
Lys Gln Glu Leu Glu Ser Met Tyr Asn Trp Asp Gly Ser Met Lys Asn
85 90 95
Leu Thr Gln Glu Met Ala Ser Ser Ile Tyr Tyr Asn Asp Tyr Ile Leu
100 105 110
Lys Pro Gly Phe Val Lys Phe Ala Asp Val Ser Pro Ala Val Thr Glu
115 120 125
Lys Leu Val Asp Ala Gly Val Asn Thr Gly Pro Ala Arg Pro Ser Arg
130 135 140
Trp Leu Gln Glu Ser Leu Asn Ala Phe Ser Arg Asn Gly Lys Asp Tyr
145 150 155 160
Pro Lys Ile Gln Val Asp Gly Lys Val Gly Ser Gly Thr Leu Ser Ala
165 170 175
Tyr Lys Ser Leu Gln Asn Lys Arg Gly Lys Val Glu Ala Cys Lys Leu
180 185 190
Ile Leu Lys Ser Leu Asp Gly Lys Gln Leu Asn Tyr Tyr Leu Ser Leu
195 200 205
Asn Met Pro Glu Tyr Thr Thr Gly Trp Ile Ala Asn Arg Ile Gly Asn
210 215 220
Val Pro Leu Glu Arg Cys Asn Glu Asp Ile Val Asn
225 230 235
<210> 61
<211> 210
<212> PRT
<213> unknown
<220>
<223> Melittin-N4gp61trunc
<400> 61
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Val Glu Gly Gly Tyr Val
20 25 30
Asn Asp Pro Lys Asp Pro Gly Gly Glu Thr Asn His Gly Val Thr Ile
35 40 45
Gln Val Ala Gln Lys His Lys Gln Glu Leu Glu Ser Met Tyr Asn Trp
50 55 60
Asp Gly Ser Met Lys Asn Leu Thr Gln Glu Met Ala Ser Ser Ile Tyr
65 70 75 80
Tyr Asn Asp Tyr Ile Leu Lys Pro Gly Phe Val Lys Phe Ala Asp Val
85 90 95
Ser Pro Ala Val Thr Glu Lys Leu Val Asp Ala Gly Val Asn Thr Gly
100 105 110
Pro Ala Arg Pro Ser Arg Trp Leu Gln Glu Ser Leu Asn Ala Phe Ser
115 120 125
Arg Asn Gly Lys Asp Tyr Pro Lys Ile Gln Val Asp Gly Lys Val Gly
130 135 140
Ser Gly Thr Leu Ser Ala Tyr Lys Ser Leu Gln Asn Lys Arg Gly Lys
145 150 155 160
Val Glu Ala Cys Lys Leu Ile Leu Lys Ser Leu Asp Gly Lys Gln Leu
165 170 175
Asn Tyr Tyr Leu Ser Leu Asn Met Pro Glu Tyr Thr Thr Gly Trp Ile
180 185 190
Ala Asn Arg Ile Gly Asn Val Pro Leu Glu Arg Cys Asn Glu Asp Ile
195 200 205
Val Asn
210
<210> 62
<211> 215
<212> PRT
<213> unknown
<220>
<223> Cecropin P1-N4gp61trunc
<400> 62
Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys
1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg Val
20 25 30
Glu Gly Gly Tyr Val Asn Asp Pro Lys Asp Pro Gly Gly Glu Thr Asn
35 40 45
His Gly Val Thr Ile Gln Val Ala Gln Lys His Lys Gln Glu Leu Glu
50 55 60
Ser Met Tyr Asn Trp Asp Gly Ser Met Lys Asn Leu Thr Gln Glu Met
65 70 75 80
Ala Ser Ser Ile Tyr Tyr Asn Asp Tyr Ile Leu Lys Pro Gly Phe Val
85 90 95
Lys Phe Ala Asp Val Ser Pro Ala Val Thr Glu Lys Leu Val Asp Ala
100 105 110
Gly Val Asn Thr Gly Pro Ala Arg Pro Ser Arg Trp Leu Gln Glu Ser
115 120 125
Leu Asn Ala Phe Ser Arg Asn Gly Lys Asp Tyr Pro Lys Ile Gln Val
130 135 140
Asp Gly Lys Val Gly Ser Gly Thr Leu Ser Ala Tyr Lys Ser Leu Gln
145 150 155 160
Asn Lys Arg Gly Lys Val Glu Ala Cys Lys Leu Ile Leu Lys Ser Leu
165 170 175
Asp Gly Lys Gln Leu Asn Tyr Tyr Leu Ser Leu Asn Met Pro Glu Tyr
180 185 190
Thr Thr Gly Trp Ile Ala Asn Arg Ile Gly Asn Val Pro Leu Glu Arg
195 200 205
Cys Asn Glu Asp Ile Val Asn
210 215
<210> 63
<211> 213
<212> PRT
<213> unknown
<220>
<223> SMAP-29-N4gp61trunc
<400> 63
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Val Glu Gly
20 25 30
Gly Tyr Val Asn Asp Pro Lys Asp Pro Gly Gly Glu Thr Asn His Gly
35 40 45
Val Thr Ile Gln Val Ala Gln Lys His Lys Gln Glu Leu Glu Ser Met
50 55 60
Tyr Asn Trp Asp Gly Ser Met Lys Asn Leu Thr Gln Glu Met Ala Ser
65 70 75 80
Ser Ile Tyr Tyr Asn Asp Tyr Ile Leu Lys Pro Gly Phe Val Lys Phe
85 90 95
Ala Asp Val Ser Pro Ala Val Thr Glu Lys Leu Val Asp Ala Gly Val
100 105 110
Asn Thr Gly Pro Ala Arg Pro Ser Arg Trp Leu Gln Glu Ser Leu Asn
115 120 125
Ala Phe Ser Arg Asn Gly Lys Asp Tyr Pro Lys Ile Gln Val Asp Gly
130 135 140
Lys Val Gly Ser Gly Thr Leu Ser Ala Tyr Lys Ser Leu Gln Asn Lys
145 150 155 160
Arg Gly Lys Val Glu Ala Cys Lys Leu Ile Leu Lys Ser Leu Asp Gly
165 170 175
Lys Gln Leu Asn Tyr Tyr Leu Ser Leu Asn Met Pro Glu Tyr Thr Thr
180 185 190
Gly Trp Ile Ala Asn Arg Ile Gly Asn Val Pro Leu Glu Arg Cys Asn
195 200 205
Glu Asp Ile Val Asn
210
<210> 64
<211> 777
<212> DNA
<213> unknown
<220>
<223> KZ144
<400> 64
aaagtattac gcaaaggcga taggggtgat gaggtatgtc aactccagac actcttaaat 60
ttatgtggct atgatgttgg aaagccagat ggtatttttg gaaataacac ctttaatcag 120
gtagttaaat ttcaaaaaga taattgtcta gatagtgatg gtattgtagg taagaatact 180
tgggctgaat tattcagtaa atattctcca cctattcctt ataaaactat ccctatgcca 240
actgcaaata aatcacgtgc agctgcaact ccagttatga atgcagtaga aaatgctact 300
ggcgttcgta gccagttgct actaacattt gcttctattg aatcagcatt cgattacgaa 360
ataaaagcta agacttcatc agctactggt tggttccaat tccttactgg aacatggaaa 420
acaatgattg aaaattatgg catgaagtat ggcgtactta ctgatccaac tggggcatta 480
cgtaaagatc cacgtataag tgctttaatg ggtgccgaac taattaaaga gaatatgaat 540
attcttcgtc ctgtccttaa acgtgaacca actgatactg atctttattt agctcacttc 600
tttgggcctg gtgcagcccg tcgtttcctg accactggcc agaatgaatt agctgctacc 660
catttcccaa aagaagctca ggcaaaccca tctatttttt ataacaaaga tgggtcacct 720
aaaaccattc aagaagttta taacttaatg gatggtaaag ttgcagcaca tagaaaa 777
<210> 65
<211> 528
<212> DNA
<213> unknown
<220>
<223> STM0016
<400> 65
aacccgatta tcgatggcat tatcgcgctg gaaggaggtt acgtctttaa tccgaaagat 60
aagggtggag caacacattg gggtattaca gaagcgacgg cacgagcaca tggttatgca 120
ggagacatgc gtgatctaac tcatgccgaa gcctacgcaa tacttgagga ggattactgg 180
atcaaaccgg gttttgatgt tatctcaacg ctgtcgtggc ctgtgagctt tgaattgtgt 240
gatgcagcgg ttaacatagg tgcataccac cctagtgcct ggttacagag atggcttaac 300
gtgttcaatc acgaaggcaa acgctatcca gacattcatg tagacggcaa cattggtccc 360
aggactttag cagccttaga acattacttg gcttggagag ggcaagaagg tgaagctgta 420
ctggtgaaag ctctgaattg cagccaaggg acctactatc taaacgtcgc tgagaagaac 480
cacaacaacg aacagttcat ctacggttgg atcaagaatc gtgtgacc 528
<210> 66
<211> 528
<212> DNA
<213> unknown
<220>
<223> Pseudin 1
<400> 66
aacccgatta tcgatggcat tatcgcgctg gaaggaggtt acgtctttaa tccgaaagat 60
aagggtggag caacacattg gggtattaca gaagcgacgg cacgagcaca tggttatgca 120
ggagacatgc gtgatctaac tcatgccgaa gcctacgcaa tacttgagga ggattactgg 180
atcaaaccgg gttttgatgt tatctcaacg ctgtcgtggc ctgtgagctt tgaattgtgt 240
gatgcagcgg ttaacatagg tgcataccac cctagtgcct ggttacagag atggcttaac 300
gtgttcaatc acgaaggcaa acgctatcca gacattcatg tagacggcaa cattggtccc 360
aggactttag cagccttaga acattacttg gcttggagag ggcaagaagg tgaagctgta 420
ctggtgaaag ctctgaattg cagccaaggg acctactatc taaacgtcgc tgagaagaac 480
cacaacaacg aacagttcat ctacggttgg atcaagaatc gtgtgacc 528
<210> 67
<211> 54
<212> DNA
<213> unknown
<220>
<223> Ranalexin
<400> 67
ttcctgggcg gtctgattgt tccagctatg atctgtgcgg tgaccaaaaa atgc 54
<210> 68
<211> 102
<212> DNA
<213> unknown
<220>
<223> Sushi 1
<400> 68
ggcttcaaac tgaaaggtat ggctcgtatc tcctgtctgc caaacggtca gtggtctaac 60
tttccaccga aatgcatccg tgaatgcgcg atggttagct ct 102
<210> 69
<211> 81
<212> DNA
<213> unknown
<220>
<223> WLBU2-Variant
<400> 69
aaacgctggg ttaaacgcgt gaaacgtgtc aaacgttggg tcaaacgtgt tgtccgtgta 60
gtgaaacgtt gggtgaaacg c 81
<210> 70
<211> 78
<212> DNA
<213> unknown
<220>
<223> Melittin
<400> 70
ggtatcggtg ctgtgctgaa agttctgacc actggtctgc cggcactgat ttcttggatc 60
aaacgcaaac gtcagcag 78
<210> 71
<211> 87
<212> DNA
<213> unknown
<220>
<223> SMAP-29
<400> 71
cgtggtctgc gtcgcctggg tcgcaaaatt gcgcacggcg tcaaaaaata cggcccgacc 60
gtgctgcgca ttatccgcat cgctggt 87
<210> 72
<211> 75
<212> DNA
<213> unknown
<220>
<223> Pleurocidin
<400> 72
ggctggggtt ctttctttaa aaaagcggct cacgttggca aacatgtagg taaagcagct 60
ctgacccact atctg 75
<210> 73
<211> 108
<212> DNA
<213> unknown
<220>
<223> Cecropin A (A. aegypti)
<400> 73
ggcggcctga aaaaactggg caaaaaactg gaaggtgccg gcaaacgtgt gttcaacgct 60
gcagaaaaag cactgccggt tgtagctggt gctaaagctc tccgtaaa 108
<210> 74
<211> 120
<212> DNA
<213> unknown
<220>
<223> Cecropin A (D. melanogaster)
<400> 74
ggctggctga aaaaaattgg caaaaaaatc gaacgcgtgg gccagcacac gcgtgatgca 60
accatccagg gtctgggtat cccacagcag gcagctaacg tagccgcgac tgctcgtggt 120
<210> 75
<211> 63
<212> DNA
<213> unknown
<220>
<223> Buforin II
<400> 75
acccgtagct ctcgtgctgg cctgcagttt ccggttggtc gcgtgcaccg tctgctccgc 60
aaa 63
<210> 76
<211> 117
<212> DNA
<213> unknown
<220>
<223> Sarcotoxin IA
<400> 76
ggatggctca aaaagattgg caagaaaatc gagcgagtcg gtcagcatac gcgtgatgca 60
actatccagg gtttaggtat cgcacagcaa gcagctaatg tagcagctac tgctcgg 117
<210> 77
<211> 294
<212> PRT
<213> unknown
<220>
<223> Pseudin 1-KZ144
<400> 77
Met Gly Leu Asn Thr Leu Lys Lys Val Phe Gln Gly Leu His Glu Ala
1 5 10 15
Ile Lys Leu Ile Asn Asn His Val Gln Gly Ser Lys Val Leu Arg Lys
20 25 30
Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu
35 40 45
Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr
50 55 60
Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp
65 70 75 80
Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser
85 90 95
Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser
100 105 110
Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly
115 120 125
Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe
130 135 140
Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln
145 150 155 160
Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys
165 170 175
Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg
180 185 190
Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile
195 200 205
Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu
210 215 220
Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly
225 230 235 240
Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn
245 250 255
Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu
260 265 270
Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu
275 280 285
His His His His His His
290
<210> 78
<211> 288
<212> PRT
<213> unknown
<220>
<223> Ranalexin-KZ144
<400> 78
Met Phe Leu Gly Gly Leu Ile Val Pro Ala Met Ile Cys Ala Val Thr
1 5 10 15
Lys Lys Cys Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu
20 25 30
Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly
35 40 45
Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys
50 55 60
Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn
65 70 75 80
Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys
85 90 95
Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro
100 105 110
Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu
115 120 125
Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala
130 135 140
Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp
145 150 155 160
Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp
165 170 175
Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly
180 185 190
Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys
195 200 205
Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro
210 215 220
Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala
225 230 235 240
Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn
245 250 255
Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp
260 265 270
Gly Lys Val Ala Ala His Arg Lys Leu Glu His His His His His His
275 280 285
<210> 79
<211> 306
<212> PRT
<213> unknown
<220>
<223> Sushi1-KZ144
<400> 79
Ala Met Gly Gly Phe Lys Leu Lys Gly Met Ala Arg Ile Ser Cys Leu
1 5 10 15
Pro Asn Gly Gln Trp Ser Asn Phe Pro Pro Lys Cys Ile Arg Glu Cys
20 25 30
Ala Met Val Ser Ser Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly
35 40 45
Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp
50 55 60
Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val
65 70 75 80
Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly
85 90 95
Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro
100 105 110
Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala
115 120 125
Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln
130 135 140
Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile
145 150 155 160
Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly
165 170 175
Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu
180 185 190
Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu
195 200 205
Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val
210 215 220
Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe
225 230 235 240
Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu
245 250 255
Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe
260 265 270
Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu
275 280 285
Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu His His His His
290 295 300
His His
305
<210> 80
<211> 297
<212> PRT
<213> unknown
<220>
<223> WLBU2-Variant-KZ144
<400> 80
Met Lys Arg Trp Val Lys Arg Val Lys Arg Val Lys Arg Trp Val Lys
1 5 10 15
Arg Val Val Arg Val Val Lys Arg Trp Val Lys Arg Gly Ser Lys Val
20 25 30
Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu
35 40 45
Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly
50 55 60
Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu
65 70 75 80
Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser
85 90 95
Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala
100 105 110
Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn
115 120 125
Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu
130 135 140
Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly
145 150 155 160
Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr
165 170 175
Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys
180 185 190
Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn
195 200 205
Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp
210 215 220
Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu
225 230 235 240
Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala
245 250 255
Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr
260 265 270
Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg
275 280 285
Lys Leu Glu His His His His His His
290 295
<210> 81
<211> 296
<212> PRT
<213> unknown
<220>
<223> Melttin-KZ144
<400> 81
Met Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala
1 5 10 15
Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly Ser Lys Val Leu
20 25 30
Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu
35 40 45
Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn
50 55 60
Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp
65 70 75 80
Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys
85 90 95
Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn
100 105 110
Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala
115 120 125
Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser
130 135 140
Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp
145 150 155 160
Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly
165 170 175
Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp
180 185 190
Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met
195 200 205
Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu
210 215 220
Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr
225 230 235 240
Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln
245 250 255
Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile
260 265 270
Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
Leu Glu His His His His His His
290 295
<210> 82
<211> 299
<212> PRT
<213> unknown
<220>
<223> SMAP-29-KZ144
<400> 82
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln
35 40 45
Thr Leu Leu Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
50 55 60
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
65 70 75 80
Cys Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
85 90 95
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
100 105 110
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
115 120 125
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
130 135 140
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
145 150 155 160
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
165 170 175
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
180 185 190
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
195 200 205
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
210 215 220
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
225 230 235 240
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
245 250 255
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
260 265 270
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
275 280 285
His Arg Lys Leu Glu His His His His His His
290 295
<210> 83
<211> 306
<212> PRT
<213> unknown
<220>
<223> Cecropin A (A.aegypti)-KZ144
<400> 83
Met Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys
1 5 10 15
Arg Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala
20 25 30
Lys Ala Leu Arg Lys Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly
35 40 45
Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr Asp
50 55 60
Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val
65 70 75 80
Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val Gly
85 90 95
Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro
100 105 110
Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala
115 120 125
Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln
130 135 140
Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile
145 150 155 160
Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly
165 170 175
Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu
180 185 190
Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu
195 200 205
Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val
210 215 220
Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe
225 230 235 240
Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu
245 250 255
Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe
260 265 270
Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu
275 280 285
Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu His His His His
290 295 300
His His
305
<210> 84
<211> 295
<212> PRT
<213> unknown
<220>
<223> Pleurocidin-KZ144
<400> 84
Met Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His Val Gly Lys His
1 5 10 15
Val Gly Lys Ala Ala Leu Thr His Tyr Leu Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 85
<211> 310
<212> PRT
<213> unknown
<220>
<223> Cecropin A (D.melanogaster)-KZ144
<400> 85
Met Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln
1 5 10 15
His Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Pro Gln Gln Ala
20 25 30
Ala Asn Val Ala Ala Thr Ala Arg Gly Gly Ser Lys Val Leu Arg Lys
35 40 45
Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu
50 55 60
Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr
65 70 75 80
Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp
85 90 95
Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser
100 105 110
Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser
115 120 125
Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly
130 135 140
Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe
145 150 155 160
Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln
165 170 175
Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys
180 185 190
Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg
195 200 205
Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile
210 215 220
Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu
225 230 235 240
Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly
245 250 255
Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn
260 265 270
Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu
275 280 285
Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu
290 295 300
His His His His His His
305 310
<210> 86
<211> 291
<212> PRT
<213> unknown
<220>
<223> BuforinII-KZ144
<400> 86
Met Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly Ser Lys Val Leu Arg Lys Gly Asp Arg
20 25 30
Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys Gly Tyr
35 40 45
Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln
50 55 60
Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly Ile Val
65 70 75 80
Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile
85 90 95
Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala
100 105 110
Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser
115 120 125
Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu
130 135 140
Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr
145 150 155 160
Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val
165 170 175
Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala
180 185 190
Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro
195 200 205
Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe
210 215 220
Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu
225 230 235 240
Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile
245 250 255
Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn
260 265 270
Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu His His His
275 280 285
His His His
290
<210> 87
<211> 309
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA-KZ144
<400> 87
Met Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln
1 5 10 15
His Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala
20 25 30
Ala Asn Val Ala Ala Thr Ala Arg Gly Ser Lys Val Leu Arg Lys Gly
35 40 45
Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu Asn Leu Cys
50 55 60
Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe
65 70 75 80
Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp Ser Asp Gly
85 90 95
Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro
100 105 110
Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg
115 120 125
Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val
130 135 140
Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp
145 150 155 160
Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe
165 170 175
Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr
180 185 190
Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile
195 200 205
Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu
210 215 220
Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala
225 230 235 240
His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln
245 250 255
Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro
260 265 270
Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val
275 280 285
Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu Glu His
290 295 300
His His His His His
305
<210> 88
<211> 226
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA-STM0016
<400> 88
Met Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln
1 5 10 15
His Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala
20 25 30
Ala Asn Val Ala Ala Thr Ala Arg Gly Ser Asn Pro Ile Ile Asp Gly
35 40 45
Ile Ile Ala Leu Glu Gly Gly Tyr Val Phe Asn Pro Lys Asp Lys Gly
50 55 60
Gly Ala Thr His Trp Gly Ile Thr Glu Ala Thr Ala Arg Ala His Gly
65 70 75 80
Tyr Ala Gly Asp Met Arg Asp Leu Thr His Ala Glu Ala Tyr Ala Ile
85 90 95
Leu Glu Glu Asp Tyr Trp Ile Lys Pro Gly Phe Asp Val Ile Ser Thr
100 105 110
Leu Ser Trp Pro Val Ser Phe Glu Leu Cys Asp Ala Ala Val Asn Ile
115 120 125
Gly Ala Tyr His Pro Ser Ala Trp Leu Gln Arg Trp Leu Asn Val Phe
130 135 140
Asn His Glu Gly Lys Arg Tyr Pro Asp Ile His Val Asp Gly Asn Ile
145 150 155 160
Gly Pro Arg Thr Leu Ala Ala Leu Glu His Tyr Leu Ala Trp Arg Gly
165 170 175
Gln Glu Gly Glu Ala Val Leu Val Lys Ala Leu Asn Cys Ser Gln Gly
180 185 190
Thr Tyr Tyr Leu Asn Val Ala Glu Lys Asn His Asn Asn Glu Gln Phe
195 200 205
Ile Tyr Gly Trp Ile Lys Asn Arg Val Thr Leu Glu His His His His
210 215 220
His His
225
<210> 89
<211> 216
<212> PRT
<213> unknown
<220>
<223> SMAP-29-STM0016
<400> 89
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Asn Pro Ile Ile Asp Gly Ile Ile Ala Leu Glu Gly Gly Tyr Val Phe
35 40 45
Asn Pro Lys Asp Lys Gly Gly Ala Thr His Trp Gly Ile Thr Glu Ala
50 55 60
Thr Ala Arg Ala His Gly Tyr Ala Gly Asp Met Arg Asp Leu Thr His
65 70 75 80
Ala Glu Ala Tyr Ala Ile Leu Glu Glu Asp Tyr Trp Ile Lys Pro Gly
85 90 95
Phe Asp Val Ile Ser Thr Leu Ser Trp Pro Val Ser Phe Glu Leu Cys
100 105 110
Asp Ala Ala Val Asn Ile Gly Ala Tyr His Pro Ser Ala Trp Leu Gln
115 120 125
Arg Trp Leu Asn Val Phe Asn His Glu Gly Lys Arg Tyr Pro Asp Ile
130 135 140
His Val Asp Gly Asn Ile Gly Pro Arg Thr Leu Ala Ala Leu Glu His
145 150 155 160
Tyr Leu Ala Trp Arg Gly Gln Glu Gly Glu Ala Val Leu Val Lys Ala
165 170 175
Leu Asn Cys Ser Gln Gly Thr Tyr Tyr Leu Asn Val Ala Glu Lys Asn
180 185 190
His Asn Asn Glu Gln Phe Ile Tyr Gly Trp Ile Lys Asn Arg Val Thr
195 200 205
Leu Glu His His His His His His
210 215
<210> 90
<211> 247
<212> PRT
<213> unknown
<220>
<223> SMAP-29-N4gp61
<400> 90
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Ala Ile Ser Lys Lys Lys Val Gly Gly Val Gly Gly Val Ile Ala Ala
35 40 45
Ile Ile Ala Ala Val Phe Ala Val Glu Gly Gly Tyr Val Asn Asp Pro
50 55 60
Lys Asp Pro Gly Gly Glu Thr Asn His Gly Val Thr Ile Gln Val Ala
65 70 75 80
Gln Lys His Lys Gln Glu Leu Glu Ser Met Tyr Asn Trp Asp Gly Ser
85 90 95
Met Lys Asn Leu Thr Gln Glu Met Ala Ser Ser Ile Tyr Tyr Asn Asp
100 105 110
Tyr Ile Leu Lys Pro Gly Phe Val Lys Phe Ala Asp Val Ser Pro Ala
115 120 125
Val Thr Glu Lys Leu Val Asp Ala Gly Val Asn Thr Gly Pro Ala Arg
130 135 140
Pro Ser Arg Trp Leu Gln Glu Ser Leu Asn Ala Phe Ser Arg Asn Gly
145 150 155 160
Lys Asp Tyr Pro Lys Ile Gln Val Asp Gly Lys Val Gly Ser Gly Thr
165 170 175
Leu Ser Ala Tyr Lys Ser Leu Gln Asn Lys Arg Gly Lys Val Glu Ala
180 185 190
Cys Lys Leu Ile Leu Lys Ser Leu Asp Gly Lys Gln Leu Asn Tyr Tyr
195 200 205
Leu Ser Leu Asn Met Pro Glu Tyr Thr Thr Gly Trp Ile Ala Asn Arg
210 215 220
Ile Gly Asn Val Pro Leu Glu Arg Cys Asn Glu Asp Ile Val Asn Leu
225 230 235 240
Glu His His His His His His
245
<210> 91
<211> 624
<212> DNA
<213> unknown
<220>
<223> N4gp61
<400> 91
atggccatct cgaagaaaaa agttggaggt gttggtggag ttattgcggc aatcattgct 60
gcagtatttg ccgttgaagg tggatacgtt aatgacccga aagatccagg aggtgaaaca 120
aaccatggtg taactattca ggtcgcacaa aaacacaagc aagaacttga gtcgatgtat 180
aactgggacg ggtcaatgaa gaatctgaca caggagatgg cctcaagtat atattacaac 240
gattatatcc tcaagcctgg ctttgtgaaa tttgcggatg taagtccagc ggttacggaa 300
aaacttgtgg atgctggagt aaatacaggt ccagcaagac caagccgttg gttacaagaa 360
tccttgaatg ctttctcacg caacggcaaa gattatccga aaatccaagt tgacgggaaa 420
gtaggttctg gaactttgag tgcttacaaa agcctgcaga ataagcgagg aaaagtggaa 480
gcctgcaaat taatactgaa gtctctggat ggcaagcagc taaactacta tctgagcctc 540
aatatgcctg agtataccac aggttggatt gcgaatcgta ttggaaatgt gcctttggaa 600
cgctgtaatg aagatatcgt caac 624
<210> 92
<211> 13
<212> PRT
<213> unknown
<220>
<223> beta 4 helix
<400> 92
Pro Asn Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr
1 5 10
<210> 93
<211> 272
<212> PRT
<213> unknown
<220>
<223> beta 4 helix:KZ144
<400> 93
Pro Asn Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Lys Val Leu
1 5 10 15
Arg Lys Gly Asp Arg Gly Asp Glu Val Cys Gln Leu Gln Thr Leu Leu
20 25 30
Asn Leu Cys Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn
35 40 45
Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Cys Leu Asp
50 55 60
Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys
65 70 75 80
Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn
85 90 95
Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala
100 105 110
Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser
115 120 125
Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp
130 135 140
Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly
145 150 155 160
Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp
165 170 175
Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met
180 185 190
Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu
195 200 205
Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr
210 215 220
Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln
225 230 235 240
Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile
245 250 255
Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
260 265 270
<210> 94
<211> 304
<212> PRT
<213> unknown
<220>
<223> beta 4 helix:gp188
<400> 94
Pro Asn Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Asn Phe Arg
1 5 10 15
Thr Lys Asn Gly Tyr Arg Asp Leu Gln Ala Leu Val Lys Glu Leu Gly
20 25 30
Leu Tyr Thr Gly Gln Ile Asp Gly Val Trp Gly Lys Gly Thr Ser Ser
35 40 45
Ser Thr Glu Thr Leu Leu Arg Gly Tyr Ala Glu Val Val Gly Lys Asn
50 55 60
Thr Gly Gly Ile Gly Leu Pro Thr Thr Ser Asp Ala Ser Gly Tyr Asn
65 70 75 80
Val Ile Thr Ala Leu Gln Arg Asn Leu Ala Phe Leu Gly Leu Tyr Ser
85 90 95
Leu Thr Val Asp Gly Ile Trp Gly Asn Gly Thr Leu Ser Gly Leu Asp
100 105 110
Lys Ala Phe Glu Val Tyr Lys Glu Arg Tyr Arg Thr Pro Thr Tyr Asp
115 120 125
Ile Ala Trp Ser Gly Lys Val Ser Pro Ala Phe Thr Ala Lys Val Lys
130 135 140
Asp Trp Cys Gly Val His Val Pro Asn His Arg Ala Pro His Trp Leu
145 150 155 160
Met Ala Cys Met Ala Phe Glu Thr Gly Gln Thr Phe Ser Pro Ser Ile
165 170 175
Lys Asn Ala Ala Gly Ser Glu Ala Tyr Gly Leu Ile Gln Phe Met Ser
180 185 190
Pro Ala Ala Asn Asp Leu Asn Val Pro Leu Ser Val Ile Arg Ser Met
195 200 205
Asp Gln Leu Thr Gln Leu Asp Leu Val Phe Lys Tyr Phe Glu Met Trp
210 215 220
Met Lys Arg Gly Lys Arg Tyr Thr Gln Leu Glu Asp Phe Tyr Leu Thr
225 230 235 240
Ile Phe His Pro Ala Ser Val Gly Lys Lys Ala Asp Glu Val Leu Phe
245 250 255
Leu Gln Gly Ser Lys Ala Tyr Leu Gln Asn Lys Gly Phe Asp Val Asp
260 265 270
Lys Asp Gly Lys Ile Thr Leu Gly Glu Ile Ser Ser Thr Leu Tyr Thr
275 280 285
Thr Tyr Tyr Lys Gly Leu Leu Pro Glu Asn Arg His Val Ile Ser Tyr
290 295 300
<210> 95
<211> 320
<212> PRT
<213> unknown
<220>
<223> SMAP-29:gp188
<400> 95
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Asn Phe Arg
20 25 30
Thr Lys Asn Gly Tyr Arg Asp Leu Gln Ala Leu Val Lys Glu Leu Gly
35 40 45
Leu Tyr Thr Gly Gln Ile Asp Gly Val Trp Gly Lys Gly Thr Ser Ser
50 55 60
Ser Thr Glu Thr Leu Leu Arg Gly Tyr Ala Glu Val Val Gly Lys Asn
65 70 75 80
Thr Gly Gly Ile Gly Leu Pro Thr Thr Ser Asp Ala Ser Gly Tyr Asn
85 90 95
Val Ile Thr Ala Leu Gln Arg Asn Leu Ala Phe Leu Gly Leu Tyr Ser
100 105 110
Leu Thr Val Asp Gly Ile Trp Gly Asn Gly Thr Leu Ser Gly Leu Asp
115 120 125
Lys Ala Phe Glu Val Tyr Lys Glu Arg Tyr Arg Thr Pro Thr Tyr Asp
130 135 140
Ile Ala Trp Ser Gly Lys Val Ser Pro Ala Phe Thr Ala Lys Val Lys
145 150 155 160
Asp Trp Cys Gly Val His Val Pro Asn His Arg Ala Pro His Trp Leu
165 170 175
Met Ala Cys Met Ala Phe Glu Thr Gly Gln Thr Phe Ser Pro Ser Ile
180 185 190
Lys Asn Ala Ala Gly Ser Glu Ala Tyr Gly Leu Ile Gln Phe Met Ser
195 200 205
Pro Ala Ala Asn Asp Leu Asn Val Pro Leu Ser Val Ile Arg Ser Met
210 215 220
Asp Gln Leu Thr Gln Leu Asp Leu Val Phe Lys Tyr Phe Glu Met Trp
225 230 235 240
Met Lys Arg Gly Lys Arg Tyr Thr Gln Leu Glu Asp Phe Tyr Leu Thr
245 250 255
Ile Phe His Pro Ala Ser Val Gly Lys Lys Ala Asp Glu Val Leu Phe
260 265 270
Leu Gln Gly Ser Lys Ala Tyr Leu Gln Asn Lys Gly Phe Asp Val Asp
275 280 285
Lys Asp Gly Lys Ile Thr Leu Gly Glu Ile Ser Ser Thr Leu Tyr Thr
290 295 300
Thr Tyr Tyr Lys Gly Leu Leu Pro Glu Asn Arg His Val Ile Ser Tyr
305 310 315 320
<210> 96
<211> 330
<212> PRT
<213> unknown
<220>
<223> Sarcotoxin IA:gp188
<400> 96
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Asn Phe Arg Thr Lys Asn Gly Tyr Arg
35 40 45
Asp Leu Gln Ala Leu Val Lys Glu Leu Gly Leu Tyr Thr Gly Gln Ile
50 55 60
Asp Gly Val Trp Gly Lys Gly Thr Ser Ser Ser Thr Glu Thr Leu Leu
65 70 75 80
Arg Gly Tyr Ala Glu Val Val Gly Lys Asn Thr Gly Gly Ile Gly Leu
85 90 95
Pro Thr Thr Ser Asp Ala Ser Gly Tyr Asn Val Ile Thr Ala Leu Gln
100 105 110
Arg Asn Leu Ala Phe Leu Gly Leu Tyr Ser Leu Thr Val Asp Gly Ile
115 120 125
Trp Gly Asn Gly Thr Leu Ser Gly Leu Asp Lys Ala Phe Glu Val Tyr
130 135 140
Lys Glu Arg Tyr Arg Thr Pro Thr Tyr Asp Ile Ala Trp Ser Gly Lys
145 150 155 160
Val Ser Pro Ala Phe Thr Ala Lys Val Lys Asp Trp Cys Gly Val His
165 170 175
Val Pro Asn His Arg Ala Pro His Trp Leu Met Ala Cys Met Ala Phe
180 185 190
Glu Thr Gly Gln Thr Phe Ser Pro Ser Ile Lys Asn Ala Ala Gly Ser
195 200 205
Glu Ala Tyr Gly Leu Ile Gln Phe Met Ser Pro Ala Ala Asn Asp Leu
210 215 220
Asn Val Pro Leu Ser Val Ile Arg Ser Met Asp Gln Leu Thr Gln Leu
225 230 235 240
Asp Leu Val Phe Lys Tyr Phe Glu Met Trp Met Lys Arg Gly Lys Arg
245 250 255
Tyr Thr Gln Leu Glu Asp Phe Tyr Leu Thr Ile Phe His Pro Ala Ser
260 265 270
Val Gly Lys Lys Ala Asp Glu Val Leu Phe Leu Gln Gly Ser Lys Ala
275 280 285
Tyr Leu Gln Asn Lys Gly Phe Asp Val Asp Lys Asp Gly Lys Ile Thr
290 295 300
Leu Gly Glu Ile Ser Ser Thr Leu Tyr Thr Thr Tyr Tyr Lys Gly Leu
305 310 315 320
Leu Pro Glu Asn Arg His Val Ile Ser Tyr
325 330
<210> 97
<211> 209
<212> PRT
<213> unknown
<220>
<223> SMAP-29:Salmonella endolysin
<400> 97
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Lys Pro Lys
20 25 30
Asp Glu Ile Phe Asp Glu Ile Leu Gly Lys Glu Gly Gly Tyr Val Asn
35 40 45
His Pro Asp Asp Lys Gly Gly Pro Thr Lys Trp Gly Ile Thr Glu Lys
50 55 60
Val Ala Arg Ala His Gly Tyr Arg Gly Asp Met Arg Asn Leu Thr Arg
65 70 75 80
Gly Gln Ala Leu Glu Ile Leu Glu Thr Asp Tyr Trp Tyr Gly Pro Arg
85 90 95
Phe Asp Arg Val Ala Lys Ala Ser Pro Asp Val Ala Ala Glu Leu Cys
100 105 110
Asp Thr Gly Val Asn Met Gly Pro Ser Val Ala Ala Lys Met Leu Gln
115 120 125
Arg Trp Leu Asn Val Phe Asn Gln Gly Gly Arg Leu Tyr Pro Asp Met
130 135 140
Asp Thr Asp Gly Arg Ile Gly Pro Arg Thr Leu Asn Ala Leu Arg Val
145 150 155 160
Tyr Leu Glu Lys Arg Gly Lys Asp Gly Glu Arg Val Leu Leu Val Ala
165 170 175
Leu Asn Cys Thr Gln Gly Glu Arg Tyr Leu Glu Leu Ala Glu Lys Arg
180 185 190
Glu Ala Asp Glu Ser Phe Val Tyr Gly Trp Met Lys Glu Arg Val Leu
195 200 205
Ile
<210> 98
<211> 303
<212> PRT
<213> unknown
<220>
<223> Pseudin 1:Acinetobacter baumannii endolysin
<400> 98
Gly Leu Asn Thr Leu Lys Lys Val Phe Gln Gly Leu His Glu Ala Ile
1 5 10 15
Lys Leu Ile Asn Asn His Val Gln Glu Tyr Asp Met Ile Leu Lys Phe
20 25 30
Gly Ser Lys Gly Asp Ala Val Ala Thr Leu Gln Lys Gln Leu Ala Lys
35 40 45
Met Gly Tyr Lys Gly Val Lys Asp Lys Pro Leu Ser Val Asp Gly His
50 55 60
Phe Gly Glu Ser Thr Glu Phe Ala Val Ile Gln Leu Gln Arg Lys Phe
65 70 75 80
Gly Leu Val Ala Asp Gly Lys Val Gly Asp Lys Thr Arg Gln Ala Leu
85 90 95
Ala Gly Asp Ser Val Ser Lys Phe Leu Lys Asp Glu Asp Tyr Lys Lys
100 105 110
Ala Ala Ile Arg Leu Lys Val Pro Glu Leu Val Ile Arg Val Phe Gly
115 120 125
Ala Val Glu Gly Leu Gly Val Gly Phe Leu Pro Asn Gly Lys Ala Lys
130 135 140
Ile Leu Phe Glu Arg His Arg Met Tyr Phe Tyr Leu Cys Gln Ala Leu
145 150 155 160
Gly Lys Thr Phe Ala Asn Ser Gln Val Lys Ile Thr Pro Asn Ile Val
165 170 175
Asn Thr Leu Thr Gly Gly Tyr Lys Gly Asp Ala Ala Glu Tyr Thr Arg
180 185 190
Leu Ser Met Ala Ile Asn Ile His Lys Glu Ser Ala Leu Met Ser Thr
195 200 205
Ser Trp Gly Gln Phe Gln Ile Met Gly Glu Asn Trp Lys Asp Leu Gly
210 215 220
Tyr Ser Ser Val Gln Glu Phe Val Asp Gln Gln Gln Leu Asn Glu Gly
225 230 235 240
Asn Gln Leu Glu Ala Phe Ile Arg Phe Ile Glu Trp Lys Pro Gly Leu
245 250 255
Leu Glu Ala Leu Arg Lys Gln Asp Trp Asp Thr Val Phe Thr Leu Tyr
260 265 270
Asn Gly Lys Asn Tyr Lys Lys Leu Gly Tyr Gln Ala Lys Phe Gln Lys
275 280 285
Glu Trp Asp His Leu Glu Pro Ile Tyr Arg Glu Lys Thr Ala Ala
290 295 300
<210> 99
<211> 308
<212> PRT
<213> unknown
<220>
<223> SMAP-29: Acinetobacter baumannii endolysin
<400> 99
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Glu Tyr Asp
20 25 30
Met Ile Leu Lys Phe Gly Ser Lys Gly Asp Ala Val Ala Thr Leu Gln
35 40 45
Lys Gln Leu Ala Lys Met Gly Tyr Lys Gly Val Lys Asp Lys Pro Leu
50 55 60
Ser Val Asp Gly His Phe Gly Glu Ser Thr Glu Phe Ala Val Ile Gln
65 70 75 80
Leu Gln Arg Lys Phe Gly Leu Val Ala Asp Gly Lys Val Gly Asp Lys
85 90 95
Thr Arg Gln Ala Leu Ala Gly Asp Ser Val Ser Lys Phe Leu Lys Asp
100 105 110
Glu Asp Tyr Lys Lys Ala Ala Ile Arg Leu Lys Val Pro Glu Leu Val
115 120 125
Ile Arg Val Phe Gly Ala Val Glu Gly Leu Gly Val Gly Phe Leu Pro
130 135 140
Asn Gly Lys Ala Lys Ile Leu Phe Glu Arg His Arg Met Tyr Phe Tyr
145 150 155 160
Leu Cys Gln Ala Leu Gly Lys Thr Phe Ala Asn Ser Gln Val Lys Ile
165 170 175
Thr Pro Asn Ile Val Asn Thr Leu Thr Gly Gly Tyr Lys Gly Asp Ala
180 185 190
Ala Glu Tyr Thr Arg Leu Ser Met Ala Ile Asn Ile His Lys Glu Ser
195 200 205
Ala Leu Met Ser Thr Ser Trp Gly Gln Phe Gln Ile Met Gly Glu Asn
210 215 220
Trp Lys Asp Leu Gly Tyr Ser Ser Val Gln Glu Phe Val Asp Gln Gln
225 230 235 240
Gln Leu Asn Glu Gly Asn Gln Leu Glu Ala Phe Ile Arg Phe Ile Glu
245 250 255
Trp Lys Pro Gly Leu Leu Glu Ala Leu Arg Lys Gln Asp Trp Asp Thr
260 265 270
Val Phe Thr Leu Tyr Asn Gly Lys Asn Tyr Lys Lys Leu Gly Tyr Gln
275 280 285
Ala Lys Phe Gln Lys Glu Trp Asp His Leu Glu Pro Ile Tyr Arg Glu
290 295 300
Lys Thr Ala Ala
305
<210> 100
<211> 313
<212> PRT
<213> unknown
<220>
<223> Sushi 1: Acinetobacter baumannii endolysin
<400> 100
Gly Phe Lys Leu Lys Gly Met Ala Arg Ile Ser Cys Leu Pro Asn Gly
1 5 10 15
Gln Trp Ser Asn Phe Pro Pro Lys Cys Ile Arg Glu Cys Ala Met Val
20 25 30
Ser Ser Glu Tyr Asp Met Ile Leu Lys Phe Gly Ser Lys Gly Asp Ala
35 40 45
Val Ala Thr Leu Gln Lys Gln Leu Ala Lys Met Gly Tyr Lys Gly Val
50 55 60
Lys Asp Lys Pro Leu Ser Val Asp Gly His Phe Gly Glu Ser Thr Glu
65 70 75 80
Phe Ala Val Ile Gln Leu Gln Arg Lys Phe Gly Leu Val Ala Asp Gly
85 90 95
Lys Val Gly Asp Lys Thr Arg Gln Ala Leu Ala Gly Asp Ser Val Ser
100 105 110
Lys Phe Leu Lys Asp Glu Asp Tyr Lys Lys Ala Ala Ile Arg Leu Lys
115 120 125
Val Pro Glu Leu Val Ile Arg Val Phe Gly Ala Val Glu Gly Leu Gly
130 135 140
Val Gly Phe Leu Pro Asn Gly Lys Ala Lys Ile Leu Phe Glu Arg His
145 150 155 160
Arg Met Tyr Phe Tyr Leu Cys Gln Ala Leu Gly Lys Thr Phe Ala Asn
165 170 175
Ser Gln Val Lys Ile Thr Pro Asn Ile Val Asn Thr Leu Thr Gly Gly
180 185 190
Tyr Lys Gly Asp Ala Ala Glu Tyr Thr Arg Leu Ser Met Ala Ile Asn
195 200 205
Ile His Lys Glu Ser Ala Leu Met Ser Thr Ser Trp Gly Gln Phe Gln
210 215 220
Ile Met Gly Glu Asn Trp Lys Asp Leu Gly Tyr Ser Ser Val Gln Glu
225 230 235 240
Phe Val Asp Gln Gln Gln Leu Asn Glu Gly Asn Gln Leu Glu Ala Phe
245 250 255
Ile Arg Phe Ile Glu Trp Lys Pro Gly Leu Leu Glu Ala Leu Arg Lys
260 265 270
Gln Asp Trp Asp Thr Val Phe Thr Leu Tyr Asn Gly Lys Asn Tyr Lys
275 280 285
Lys Leu Gly Tyr Gln Ala Lys Phe Gln Lys Glu Trp Asp His Leu Glu
290 295 300
Pro Ile Tyr Arg Glu Lys Thr Ala Ala
305 310
Claims (25)
- 그람-음성 박테리아의 세포벽을 분해하는 활성을 갖는 효소 및 N-말단 또는 C-말단 또는 양 말단에서 상기 효소에 융합된 펩티드 신장(peptide stretch)으로 구성된 융합 단백질로서,
상기 펩티드 신장이 양친매성 펩티드, 스시 펩티드, 디펜신, 소수성 펩티드 또는 항균성 펩티드인, 융합 단백질. - 제1항에 있어서,
상기 융합 단백질이 SEQ ID NO: 63 내지 90에 따른 아미노산 서열을 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항 또는 제2항에 있어서,
상기 융합 단백질이 N-말단에 부가적인 아미노산 잔기를 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항 내지 제3항 중 어느 한 항에 있어서,
상기 융합 단백질이 C-말단 및/또는 N-말단에 태그 또는 부가적인 단백질을 포함하는 것을 특징으로 하는, 융합 단백질. - 제4항에 있어서,
상기 태그 또는 부가적인 단백질이 하나 이상의 부가적인 아미노산 잔기에 의해 융합 단백질에 연결되는 것을 특징으로 하는, 융합 단백질. - 제1항 내지 제5항 중 어느 한 항에 있어서,
상기 펩티드 신장이 하나 이상의 부가적인 아미노산 잔기에 의해 융합 단백질에 연결되는 것을 특징으로 하는, 융합 단백질. - 제1항에 있어서,
상기 효소가 엔돌리신, 오토리신 또는 박테리오신인 것을 특징으로 하는, 융합 단백질. - 제7항에 있어서,
상기 효소가 SEQ ID NO: 22 내지 25에 따른 아미노산 서열을 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항에 있어서,
상기 항균성 펩티드가 SEQ ID NO: 6 내지 16 또는 26 내지 31에 따른 아미노산 서열을 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항에 있어서,
상기 스시 펩티드가 SEQ ID NO: 32에 따른 아미노산 서열을 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항에 있어서,
상기 소수성 펩티드가 SEQ ID NO: 28, 33 또는 35에 따른 아미노산 서열을 나타내는 것을 특징으로 하는, 융합 단백질. - 제1항 내지 제11항 중 어느 한 항에 있어서,
상기 그람-음성 박테리아가 하기로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 융합 단백질:
엔테로박테리아세아에(Enterobacteriaceae),
특히 에스케리챠(Escherichia), 살모넬라(Salmonella), 쉬겔라(Shigella), 시트로박테르(Citrobacter), 에드워드시엘라(Edwardsiella), 엔테로박테르(Enterobacter), 하프니아(Hafnia), 클레브시엘라(Klebsiella), 모르가넬라(Morganella), 프로테우스(Proteus), 프로비덴시아(Providencia), 세라티아(Serratia) 및 예르시니아(Yersinia),
슈도모나다세아에(Pseudomonadaceae),
특히 슈도모나스(Pseudomonas), 부르크홀데리아(Burkholderia), 스테노트로포모나스(Stenotrophomonas), 쉐와넬라(Shewanella), 스핑고모나스(Sphingomonas) 및 코마모나스(Comamonas),
네이세리아(Neisseria), 모라셀라(Moraxella), 비브리오(Vibrio), 아에로모나스(Aeromonas), 브루셀라(Brucella), 프란시셀라(Francisella), 보르데텔라(Bordetella), 레지오넬라(Legionella), 바르토넬라(Bartonella), 콕시엘라(Coxiella), 헤모필러스(Haemophilus), 파스퉤렐라(Pasteurella), 만헤이미아(Mannheimia), 악티노바실러스(Actinobacillus), 가르드네렐라(Gardnerella), 스피로카에타세아에(Spirochaetaceae),
특히 트레포네마(Treponema) 및 보렐리아(Borrelia),
렙토스피라세아에(Leptospiraceae), 캄필로박테르(Campylobacter), 헬리코박테르(Helicobacter), 스필릴룸(Spirillum), 스트렙토바실러스(Streptobacillus), 박테로이다세아에(Bacteroidaceae),
특히 박테로이데스(Bacteroides), 푸소박테리움(Fusobacterium), 프레보텔라(Prevotella) 및 포르피로모나스(Porphyromonas), 및
아시네토박테르(Acinetobacter),
특히 A. 바우마니이(A. baumanii). - 제1항에 있어서,
상기 양친매성 펩티드가 발린, 이소류신, 류신, 메티오닌, 페닐알라닌, 트립토판, 시스테인, 알라닌, 티로신, 히스티딘, 트레오닌, 세린, 프롤린 및 글리신 잔기로 이루어진 군으로부터 선택되는 하나 이상의 소수성 아미노산 잔기와 조합된, 리신, 아르기닌 및 히스티딘 잔기로 이루어진 군으로부터 선택되는 하나 이상의 양전하 아미노산 잔기를 포함하는 것을 특징으로 하는, 융합 단백질. - 제1항에 있어서,
상기 양친매성 펩티드 내 상기 아미노산 잔기의 약 70% 이상이 아르기닌 또는 리신 잔기이고, 상기 양친매성 펩티드 내 상기 아미노산 잔기의 약 30% 이상이 발린, 이소류신, 류신, 메티오닌, 페닐알라닌, 트립토판, 시스테인, 알라닌, 티로신, 히스티딘, 트레오닌, 세린, 프롤린 또는 글리신 잔기인 것을 특징으로 하는, 융합 단백질. - 제1항 내지 제14항 중 어느 한 항에 있어서,
상기 펩티드 신장이 약 5 내지 약 100 개의 아미노산 잔기, 특히 약 5 내지 50 개의 아미노산 잔기, 특히 약 5 내지 30 개의 아미노산 잔기를 포함하는 것을 특징으로 하는, 융합 단백질. - 제1항 내지 제15항 중 어느 한 항에 따른 융합 단백질을 코딩하는, 단리된 핵산 분자.
- 제16항에 따른 핵산 분자를 포함하는 벡터.
- 제16항에 따른 핵산 분자 또는 제17항에 따른 벡터를 포함하는 숙주 세포.
- 제18항에 있어서,
상기 세포가 박테리아 세포 또는 효모 세포인 것을 특징으로 하는, 숙주 세포. - 제1항 내지 제15항 중 어느 한 항에 있어서,
의약, 진단 수단 또는 화장품 성분으로서 사용하기 위한, 융합 단백질. - 제1항 내지 제15항 중 어느 한 항에 있어서,
그람-음성 박테리아 감염의 치료 또는 예방용 의약으로서 사용하기 위한, 융합 단백질. - 제1항 내지 제15항 중 어느 한 항에 있어서,
살균제로서 사용하기 위한, 융합 단백질. - 식료품, 식품 가공 장비, 식품 가공 공장, 식료품과 접촉하게 되는 표면, 의료 장치, 병원 및 수술기기 표면의 그람-음성 박테리아 오염의 치료 또는 예방을 위한, 제1항 내지 제15항 중 어느 한 항에 따른 융합 단백질의 용도.
- 의료적, 식품 또는 사료 또는 환경적 진단에서 진단 수단으로서 사용하기 위한, 제1항 내지 제15항 중 어느 한 항에 따른 융합 단백질의 용도.
- 제1항 내지 제15항 중 어느 한 항에 따른 융합 단백질을 포함하는 약학 조성물.
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