励途FB-110X设备实用案例

An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins

Zhenhua Tian1,5, Peng Sun1,2,5, Yan Yan1,5, Zhuhua Wu1, Qingfei Zheng1, Shuaixiang Zhou1, Hua Zhang1, Futao Yu1, Xinying Jia1, Dandan Chen3, Attila Mándi4, Tibor Kurtán4 & Wen Liu1,3*

Protein expression and purification. The genes pyrE3 and pyrI4 from S. rugosporus and the genes chlE3 and chlL from S. antibioticus were individually amplified by PCR using the corresponding primers, the sequences of which are presented in Supplementary Table 2. These PCR products were purified, digested with NdeI and HindIII (or NdeI and XhoI) and then ligated into the vector pET28a(+), which was digested with the same enzymes. The resulting recombinant plasmids, which are listed in Supplementary Table 1, were transferred into E. coli BL21(DE3) for protein overexpression. The culture of each E. coli transformant was incubated in Luria-Bertani (LB) medium containing 50 μg/ml kanamycin at 37 °C and at 250 r.p.m. until the cell density reached 0.6–0.8 at OD600. Protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, followed by further incubation for 40 h at 16 °C. The cells were harvested by centrifuging at 5,000 r.p.m. for 20 min at 4 °C and were resuspended in 30 ml of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH 7.0). After disruption by FB-110X Low Temperature Ultra-pressure Continuous Flow Cell Disrupter (Shanghai Litu Mechanical Equipment Engineering Co., Ltd, China), the soluble fraction was collected and subjected to purification of each target protein using a HisTrap FF column (GE Healthcare, USA). The desired protein fractions, as determined by SDS-PAGE, were concentrated and desalted using a PD-10 Desalting Column (GE Healthcare, USA) according to the manufacturer’s protocols. The concentration of protein was determined by Bradford assay using bovine serum albumin (BSA) as the standard.

蛋白表达和纯化。基因pyrE3 pyrI4从美国rugosporus和基因chlE3 chlL从美国antibioticus单独放大使用对应的引物,通过PCR序列补充表2中给出。这些PCR产物纯化,消化与心好HindIII(或心好和XhoI),然后结扎成向量pET28a(+),这是与同一酶消化。产生的重组质粒,补充表1中列出的反式¬转移到大肠杆菌BL21(DE3)蛋白质过度。每个大肠杆菌转化株的文化在Luria-Bertani孵化(磅)中包含50μg /毫升卡那霉素在37°C和250 r.p.m.直到OD600细胞密度达到0.6 - -0.8。蛋白表达是诱导的isopropyl-β-D-thiogalactopyranoside(IPTG)醉后一个0.2毫米的浓度,符合¬低下通过进一步孵化为40小时16°C。细胞收获了离心法在5000 r.p.m. 20分钟在4°C和resuspended 30毫升的裂解缓冲(50毫米Tris-HCl,300毫米氯化钠,10%的甘油,pH值7.0)。使用fb - 110 x低温下连续流动孔隙细胞分裂者(上海励途机械设备工程有限公司,中国),可溶性分数收集并受净化的每个目标蛋白质使用HisTrap FF列(美国通用电气医疗集团)。所需的蛋白质分数,由sds - page、集中和脱盐使用PD-10脱盐列(美国通用电气医疗集团)根据开发¬真正的协议。蛋白质的浓度是由布拉德福德化验使用牛血清白蛋白(BSA)作为标准。