为促进重组藻蓝蛋白的胞外分泌,该研究探索温度、诱导剂[异丙基-β-D-硫代半乳糖苷(isoprpyl-β-D-thiogalatopyranoside,IPTG)和乳糖],以及培养基添加剂(甘氨酸、Triton X-100、十二烷基硫酸钠和Tween-80)对大肠杆菌胞外分泌重组螺旋藻荧光藻蓝蛋白胞β亚基(β-subunit of C-phycocyanin,CpcB)荧光强度的影响。结果表明,在25℃的条件下,重组体系表达24 h胞外培养基具有明显的CpcB荧光,且荧光强度随着表达时间的增加而逐步升高,温度是影响胞外分泌的关键因素。添加乳糖可显著增加胞外培养基CpcB的荧光强度,当用10 g/L的乳糖作为诱导剂时,荧光强度达到最大值,比未加诱导剂高5.1倍;而用IPTG作诱导剂,培养基没有明显的CpcB荧光。添加25 g/L的甘氨酸可使得胞外CpcB的荧光强度提高6.3倍,添加1.5% (体积分数)的Tween-80也可使其荧光强度提高2.5倍。荧光藻蓝蛋白的胞外分泌为改善其异源生产的效率提供便利。
In order to promote the extracellular secretion of phycocyanin, the effects of temperature, inducers (IPTG and lactose) and medium additives (glycine, Triton X-100, sodium dodecyl sulfate and Tween-80) on the fluorescence intensity of extracellular β-subunit C-phycocyanin (CpcB) of Spirulina secreted by E. coli were investigated. The results showed that the extracellular medium exhibited an apparent CpcB-derived fluorescence after the recombinant expression for 24 h at 25℃. The fluorescence intensity gradually increased in a time-dependent manner. Temperature was the key factor affecting the extracellular secretion. Lactose induction could significantly increase the fluorescence intensity of the extracellular CpcB, which reached the highest, about 5.1 times higher than that of the control, with the lactose concentration of 10 g/L. Comparably, the addition of IPTG had no effect on the fluorescence intensity. The extracellular fluorescence intensity increased 6.3 times with the addition of 25 g/L glycine in the medium, and 2.5 times when 1.5% of φ(Tween-80) was added. The extracellular secretion caused by altering culture conditions and supplementing additives facilitates the heterologous production of fluorescent phycocyanin in E. coli.
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