CAJ | 학술논문

目的:构建硫氧还蛋白-(His)6-瑞替普酶融合表达载体,提高瑞替普酶在大肠杆菌中的表达量,简化rPA的分离纯化.方法:将rPA基因克隆于原核表达载体pET32a硫氧还蛋白-(组氨酸)6标签下游,在大肠杆菌BL21(DE3)中用乳糖进行诱导表达.采用一步稀释法对融合蛋白体外复性后,使用Ni2+亲和层析柱,对包涵体复性液进行初步纯化.采用体外溶圈法对复性后和纯化后的融合蛋白进行生物活性的测定.结果:得到分子量约为56 kDa的融合蛋白,表达量达到总蛋白的30%以上.比本实验室构建的非融合表达载体表达量提高了约50%.经过一步Ni2+亲和层析,融合蛋白纯度达到80%以上.体外溶圈法实验表明融合蛋白复性后和纯化后具有溶栓活性.结论:与非融合表达相比,融合表达的表达量明显提高.即使N端额外融合一段融合多肽,rPA仍然具有生物学活性.
목적:구건류양환단백-(His)6-서체보매융합표체재체,제고서체보매재대장간균중적표체량,간화rPA적분리순화.방법:장rPA기인극륭우원핵표체재체pET32a류양환단백-(조안산)6표첨하유,재대장간균BL21(DE3)중용유당진행유도표체.채용일보희석법대융합단백체외복성후,사용Ni2+친화층석주,대포함체복성액진행초보순화.채용체외용권법대복성후화순화후적융합단백진행생물활성적측정.결과:득도분자량약위56 kDa적융합단백,표체량체도총단백적30%이상.비본실험실구건적비융합표체재체표체량제고료약50%.경과일보Ni2+친화층석,융합단백순도체도80%이상.체외용권법실험표명융합단백복성후화순화후구유용전활성.결론:여비융합표체상비,융합표체적표체량명현제고.즉사N단액외융합일단융합다태,rPA잉연구유생물학활성.
AIM:To increase the expression amount of reteplase,a vector was constructed which expressed the fusion protein thioredoxin-(His)6-rPA. The purification of reteplase was simplified by one-step affinity chromatography in this study. METHOD: Reteplase gene was inserted into the prokaryotic expression vector pET32a and was fused to the downstream of the thioredoxin and the (His)6Tag in the same reading frame. The fusion protein was expressed in E. coli BL21 (DE3) induced by lactose. After refolded in vitro by one-step dilution,the fusion protein was simply purified using Ni2+-chelating chromatography. Fibrin plate assay was used to detect the bioactivity of the fusion protein. RESULT:A fusion protein of 56 kD was obtained. The amount of the fusion protein was more than 60% of the total bacterial protein. Comparing with the non-fusion reteplase,the yields of target protein increased about 50%. The purity of the fusion protein reached above 80% after one-step purification using Ni2+-chelating chromatography. The bioactivity of the refolded and purified fusion protein was detected. CONCLUSION: Expression level of the fusion protein increased distinctively compared with that of the non-fusion protein. Bioactivity of reteplase was detected even though a fusion peptide was fused to the N-terminal of reteplase.