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.

蛋白表達(dá)和純化?；?/font>pyrE3 pyrI4從美國(guó)rugosporus和基因chlE3 chlL從美國(guó)antibioticus單獨(dú)放大使用對(duì)應(yīng)的引物,通過(guò)PCR序列補(bǔ)充表2中給出。這些PCR產(chǎn)物純化,消化與心好HindIII(或心好和XhoI),然后結(jié)扎成向量pET28a(+),這是與同一酶消化。產(chǎn)生的重組質(zhì)粒,補(bǔ)充表1中列出的反式¬轉(zhuǎn)移到大腸桿菌BL21(DE3)蛋白質(zhì)過(guò)度。每個(gè)大腸桿菌轉(zhuǎn)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細(xì)胞密度達(dá)到0.6 - -0.8。蛋白表達(dá)是誘導(dǎo)的isopropyl-β-D-thiogalactopyranoside(IPTG)醉后一個(gè)0.2毫米的濃度,符合¬低下通過(guò)進(jìn)一步孵化為40小時(shí)16°C。細(xì)胞收獲了離心法在5000 r.p.m. 20分鐘在4°C和resuspended 30毫升的裂解緩沖(50毫米Tris-HCl,300毫米氯化鈉,10%的甘油,pH值7.0)。使用fb - 110 x低溫下連續(xù)流動(dòng)孔隙細(xì)胞分裂者(上海勵(lì)途機(jī)械設(shè)備工程有限公司,中國(guó)),可溶性分?jǐn)?shù)收集并受凈化的每個(gè)目標(biāo)蛋白質(zhì)使用HisTrap FF列(美國(guó)通用電氣醫(yī)療集團(tuán))。所需的蛋白質(zhì)分?jǐn)?shù),由sds - page、集中和脫鹽使用PD-10脫鹽列(美國(guó)通用電氣醫(yī)療集團(tuán))根據(jù)開(kāi)發(fā)¬真正的協(xié)議。蛋白質(zhì)的濃度是由布拉德福德化驗(yàn)使用牛血清白蛋白(BSA)作為標(biāo)準(zhǔn)。