조직형 플라스미노겐 액티베이터와 관련 변이 단백질들을 발현하는 알팔파 형질전환체
Expression of Tissue-Type Plasminogen Activator and Its Derivative Proteins in Transgenic Alfalfa Plants
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의료용 단백질로 중요한 인체 혈관 내피세포 유래 t-PA를 알팔 파 식물체를 이용하여 생산하는 기술을 개발하였다. 식물체에서 발현된 t-PA는 동물세포에서 생산된 t-PA와 동등한 시험관 내의 인 공 혈전 용해 활성 및 생화학적 특성에서 유사함을 가지고 있었다. 본 연구에서는 식물체에서 발현되는 t-PA 단백질의 알팔파 코돈 이용에 최적화된 합성 유전자이용 양적 증대, 소포체에 표적에 따 른 발현양 증대, 6개의 histidine의 부착에 따른 정제의 효율성을 고 려하였고 두 종류의 프로모터 (CaMV 35S와 알팔파 Rbcsk-1A)를 이용하여 t-PA 및 파생 단백질들의 발현 효율을 비교 확인하고자 6가지의 식물발현 벡터를 제작하였다. 0.3%의 제초제 살포 후에 저항성을 갖는 알팔파 식물체들의 t-PA 유전자 및 파생 유전자의 genomic DNA내의 삽입 유무는 PCR법을 활용하여 t-PA, 파생 유전 자 및 합성 유전자의 크기에 해당하는 1.6 kb의 PCR 산물을 확인 할 수 있었다. 6가지 발현벡터로 형질전환된 알팔파 잎 추출물에서 전체 수용성 단백질내의 t-PA 및 파생 단백질의 평균 발현양은 9.7-39.5 μg/TSP (mg)로 측정되었다. 이중 p221a-t-PAER 발현벡터 로 형질전환된 알팔파의 잎 추출물에서 가장 높은 75.1 μg/TSP (mg)의 발현양을 나타났다. 알팔파 잎에 발현된 재조합 t-PA 분자 량은 상업적으로 판매되는 t-PA와 동일한 68 kDa으로 확인되었다. 형질전환된 알팔파 잎 추출물들의 평균 피브린 용해활성은 3.2-8.1 cm를 나타내었다. 또한 t-PA 및 그의 파생 단백질을 발현하는 알팔 파 식물체는 야생종 알팔파와 비교했을 때 성장에 있어서 별 다른 차이를 보이지 않았다.
Abstract
Tissue-type plasminogen activator (t-PA) is a thrombolytic agent important in fibirn clot lysis. T-PA causes fibirn- specific plasminogen activation. Six binary vectors harboring t-PA and its derivative genes were cloned and expressed in transgenic alfalfa plants. The insertion of the t-PA and its derivative genes in genomic DNA of alfalfa plants was confirmed by PCR. The presence of the t-PA and its derivative transcripts in total RNAs of the transgenic alfalfa leaves was verified by RT-PCR. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was 75.1 µg/ total soluble protein (mg) in alfalfa plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 9.7 to 39.5 µg/ total soluble proteins (mg). Western blot analysis of the transformed alfalfa leaves revealed bands of approximately 68-kDa recombinant t-PA and its derivative proteins. The fibrinolysis of recombinant t-PA and its derivative proteins was confirmed by a fibrin plate assay (range from 3.2 to 8.1 cm). The results presented provide information for the development of an additional production of recombinant human proteins having pharmaceutical applications using transgenic plants.
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