一种生物活性葡萄激酶在毕赤酵母中的高水平表达

H. Faraji, M. Ramezani, H. Sadeghnia, K. Abnous, F. Soltani, B. Mashkani
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引用次数: 11

摘要

葡萄激酶(Staphylokinase, SAK)作为第三代溶栓分子,是治疗血栓形成的一种很有前景的药物。SAKфC的SAK变体在毕赤酵母菌株KM71H和GS115中表达。SAK的密码子适应指数由0.75提高到0.89。重组SAK (rSAK)的表达量在3%甲醇刺激48小时后达到最大值(310 mg/L培养基),并保持稳定至第5天。该酶在pH 8.6、37℃条件下活性最高。在温度20 ~ 37℃,pH值6.8 ~ 9范围内具有较高的活性(相对残留活性大于80%)。结果表明,ppastoris KM71H向培养基中分泌的rSAK蛋白占总蛋白的73.8%。纯化蛋白和非纯化蛋白的rSAK比活性分别为9002 U/mg和21042 U/mg。纯化蛋白的纯度为720µg/mL,纯化系数为2.34,纯度大于99%。Western blot分析显示两个条带接近22和18.6 kDa。结果表明,酵母培养上清液中具有较高的表达量和较低的蛋白杂质,是表达具有生物活性且无内毒素的rSAK蛋白的适宜宿主。
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High-level expression of a biologically active staphylokinase in Pichia pastoris
ABSTRACT Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20–37°C and pH ranges of 6.8–9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042 U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.
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