Zhou Yu-Xun, Cao Wei, Wei Dong-Zhi, Luo Qing-Ping, Wang Jin-Zhi
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Jul;21(4):615-21.
33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.
33个氨基酸的抗生素肽腺调节素（ADR）最初是从南美洲树蛙双色叶泡蛙的皮肤中分离出来的，它在非极性介质中形成α-螺旋两亲结构，具有广谱抗菌活性和高效的裂解能力。Adr基因被克隆到pET32a中，并转化到大肠杆菌BL21(DE3)中。对大肠杆菌BL21(DE3)/pET32a-adr的培养和诱导条件进行了优化。研究了诱导时间点、培养物中IPTG浓度和诱导时间这三个因素对Trx-ADR表达水平的影响。结果表明，诱导时间点对表达水平的影响最为显著。测试了9种培养和诱导BL21(DE3)/pET32a-adr的培养基，以实现目标蛋白的高表达水平。发现培养基中的葡萄糖在维持Trx-ADR的稳定和高表达水平方面起着重要作用。最佳诱导条件如下：培养基为2×YT + 0.5%葡萄糖，诱导时间点为OD600 = 0.9，培养物中IPTG的终浓度为0.1 mmol/L，诱导时间为4小时。按照前期恒pH、后期指数补料的策略培养BL21(DE3)/pET32a-adr以获得高细胞密度。在整个补料分批培养阶段，通过控制葡萄糖的补料，将培养物的比生长速率控制在约每小时0.15，将乙酸的积累控制在低水平（<2 g/L），但质粒稳定性不能很好地维持。培养结束时，培养物中40%的细菌失去了质粒。结果，目标蛋白的表达水平急剧下降，但90%的Trx-ADR为可溶形式。表达的融合蛋白没有抗菌活性，而从Trx-ADR中裂解出来的天然形式的ADR具有明显的抗菌活性。
