[건국대학교] 의생명공학과 - 단백질공학 리포트(단백질조사)

본 내용은 " [건국대학교] 의생명공학과 - 단백질공학 리포트(단백질조사) " 의 원문 자료에서 일부 인용된 것입니다. 2024.05.27

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• 1. Tissue Plasminogen Activator (tPA)

  Tissue Plasminogen Activator (tPA)는 혈중 plasminogen을 활성화하여 혈전을 용해시키는 plasmin으로 전환하는 과정을 촉진시키는 단백질(효소)입니다. Plasmin은 혈전을 분해하는 데 중요한 역할을 합니다. tPA는 주로 혈전성 질환 치료에 사용되는 혈전 용해제로, 혈관 내피세포에서 발견됩니다. 사람의 tPA는 약 70kDa의 분자량을 가지고 있습니다. tPA는 허혈성 뇌졸중, 급성 심근 경색, 급성 중증 폐동맥 색전증, 말초 동맥 혈전증 등을 치료하는 데 사용되지만, 출혈성 뇌졸중에는 사용할 수 없습니다. tPA를 대량 생산하기 위해서는 eukaryotic 세포를 이용해야 하지만, 이는 상대적으로 느린 증식 속도로 인해 어려움이 있습니다. 또한 tPA의 반감기가 짧아 지속적인 투여가 필요한 단점이 있습니다.

• 2. 재조합 tPA (rtPA)

  tPA는 DNA 재조합 기술을 이용해 1982년 Genetech에서 처음 생산되었으며, 이를 rtPA라고 합니다. 재조합 방법은 다음과 같습니다. 포유 동물 조직에서 DNA 템플릿을 분리하고, mRNA를 합성한 후 reverse transcriptase를 이용해 cDNA 라이브러리를 만듭니다. 이는 진핵세포의 DNA를 사용하므로 인트론을 제거하기 위함입니다. 이후 cDNA를 벡터 플라스미드에 클로닝하고 대장균을 최초 host 세포로 이용해 발현시킵니다. Methotrexate를 이용한 형질 전환으로 더 많은 재조합 단백질을 생산할 수 있게 되었습니다. 하지만 사람의 tPA는 PTM이 필요하므로, 포유류 세포나 CHO 세포를 사용해 의약품 tPA를 생산하였습니다.

• 3. 변형된 tPA 구조

  재조합 기술을 통해 의약적으로 이용되는 tPA 변형체에는 Alteplase, Reteplase, Tenecteplase 등이 있습니다. Alteplase와 Reteplase의 화학식은 다르지만 구조는 유사합니다. Reteplase는 인간 tPA의 527개 아미노산 중 355개를 포함하며, kringle-2와 serine protase domain을 가지고 있습니다. 또한 retavase-kringle-1, finger, EGF 도메인이 삭제된 특성을 가지고 있습니다.

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• 1. Tissue Plasminogen Activator (tPA)

  Tissue Plasminogen Activator (tPA) is a serine protease enzyme that plays a crucial role in the dissolution of blood clots, making it an important therapeutic agent in the treatment of various cardiovascular and cerebrovascular diseases. tPA is naturally produced by the endothelial cells lining the blood vessels and is responsible for converting the inactive zymogen plasminogen into the active enzyme plasmin, which then breaks down fibrin, the main component of blood clots. The use of tPA as a thrombolytic agent has been extensively studied and has proven to be effective in the treatment of acute ischemic stroke, myocardial infarction, and other thrombotic conditions. However, the clinical application of tPA is not without its challenges, as it carries a risk of bleeding complications and has a relatively short half-life in the body. Ongoing research is focused on improving the safety and efficacy of tPA-based therapies, as well as exploring alternative strategies for clot dissolution and prevention.

• 2. 재조합 tPA (rtPA)

  Recombinant tissue plasminogen activator (rtPA) is a genetically engineered version of the naturally occurring tPA, which has been developed to overcome some of the limitations of the native enzyme. The production of rtPA involves the use of recombinant DNA technology, where the gene encoding for tPA is inserted into a host cell, typically a mammalian cell line, which then produces the recombinant protein. Compared to the natural tPA, rtPA has several advantages, including a longer half-life in the body, improved stability, and potentially reduced risk of bleeding complications. The use of rtPA has become the standard of care for the treatment of acute ischemic stroke, as it has been shown to be effective in restoring blood flow and improving clinical outcomes when administered within a specific time window. However, the use of rtPA is still associated with some risks, and ongoing research is focused on developing even more effective and safer thrombolytic agents. The continued advancement of rtPA and other thrombolytic therapies is crucial for improving the management of various cardiovascular and cerebrovascular diseases.

• 3. 변형된 tPA 구조

  The structure of tissue plasminogen activator (tPA) has been extensively studied, and various modifications to the native tPA structure have been explored to enhance its therapeutic potential. One of the key approaches has been the development of modified or engineered tPA variants that aim to improve specific properties, such as increased clot selectivity, reduced bleeding risk, and extended half-life in the body. Some examples of modified tPA structures include: 1. Mutant tPA: Specific amino acid substitutions in the tPA sequence can lead to altered enzymatic activity, substrate specificity, or pharmacokinetic properties. 2. Pegylated tPA: The attachment of polyethylene glycol (PEG) molecules to tPA can increase its circulatory half-life and reduce its clearance rate. 3. Chimeric tPA: Combining tPA with other proteins or domains, such as fibrin-binding moieties, can enhance the clot-targeting ability and specificity of the thrombolytic agent. 4. Nanoparticle-encapsulated tPA: Encapsulating tPA within nanoparticles can protect the enzyme from degradation, improve its delivery to the target site, and potentially reduce systemic side effects. These structural modifications have shown promising results in preclinical and clinical studies, demonstrating the potential to improve the safety and efficacy of tPA-based therapies. Ongoing research in this field continues to explore innovative approaches to optimize the therapeutic potential of tPA and develop even more effective and targeted thrombolytic agents for the management of various cardiovascular and cerebrovascular diseases.