实验 7 外源基因在大肠杆菌中的表达(或诱导表达)和检测 (1)

显色反应： 在1ml样品（细胞与培养基混合液）中直接加入 X-Gal 200ul. 充分混合，观察颜色反应
（如果时间紧张，将三个样品带回，观察、拍照！）
Introduction of pRSET Vector The pRSET vectors are pUC-derived expression vectors designed for high-level protein expression and purification from cloned genes in E. coli. High levels of expression of DNA sequences cloned into the pRSET vectors are made possible by the presence of the T7 promoter. In addition, DNA inserts are positioned downstream and in frame with a sequence that encodes an N-terminal fusion peptide. This sequence includes an ATG translation initiation codon, a polyhistidine tag that functions as a metal binding domain in the translated protein, a transcript stabilizing sequence from gene 10 of phage T7, the Xpress. epitope, and the enterokinase cleavage recognition sequence.
T7
激活 activation
Gene
启动转录 start to transcript
目的基因插入表达载体 cloned into vector
Pilot expression
转化入特定菌株，挑单菌斑培养
screening
25ml SOB
37
Inoculate OD600 = 0.1
shaking
实验七
外源基因在大肠杆菌中的表达 （或诱导表达）和检测
一、【实验原理】
将外源基因克隆在含有T7启动子的表达载体中，让 其在E. coli中表达。先让宿主菌生长，lacI产生的阻遏蛋 白与lac操纵基因结合，从而抑制T7 RNA聚合酶的产 生，使外源基因不能转录及表达。然后向培养基中加入 lac操纵子的诱导物IPTG（异丙基硫代-β-D-半乳 糖），阻遏蛋白不能与操纵基因结合，启动T7 RNA聚 合酶的产生，从而诱导外源基因大量转录并高效表达。 表达蛋白可经显色反应或聚丙烯凝胶电泳SDS-PAGE检 测分析。
原核（大肠杆菌）表达系统
特点： 表达质粒独立运转表达 基因重组速度快，生产工艺简单， 成本低，表达量较高， 重组蛋白翻译后修饰较差。
1978年，人胰岛素insulin 基因首次表达 目前仍是一个重要的表达系统
应用：活性要求不高的蛋白质 如抗原制备
原核表达系统－E.coli:
transformation
Lac 来源于乳糖操纵子的启动子
T7 来源于噬菌体的启动子
PL 噬菌体早期启动子
诱导表达 expression by induction IPTG: isopropylthio-β-D-thiogalactoside
异丙基硫代-β–D-半乳糖苷 lactose analog
T7
Gene
IPTG
诱导产生T7 RNA polymerase (T7 RNP)
8. Repeat this freeze-thaw two to three additional times and pellet the insoluble protein in a microcentrifuge for 10 minutes at maximum speed at +4°C. (9, 10 不做， 改为显色反应！) 9. Remove the supernatant to a fresh labeled tube. To 100 µl of supernatant sample, add an equal volume of 2X SDSPAGE sample buffer. Resuspend the pellet in 100 µl of 1X SDS-PAGE sample buffer. 10. Load 10-20 µl of each of the supernatant and pellet samples after boiling for 5 minutes on an appropriate SDSPAGE gel and electrophorese.
7. When all time points have been collected, resuspend each pellet in 100 µl of 20 mM phosphate buffer at neutral pH, and freeze in liquid nitrogen or methanol/dry ice (exercise caution when handling liquid nitrogen, it can cause severe burns if it comes in contact with the skin, wear appropriate protective equipment). Thaw the frozen lysate at ቤተ መጻሕፍቲ ባይዱ2°C.
二、【实验目的】
通过本实验了解外源基因在原核细胞中表达的 特点和方法。要求掌握： 1、什么是基因表达？掌握原核表达系统的工作原理 2、根据pRSET质粒的特点，说明为什么需要诱导表 达？其原理是什么？ 3、掌握表达产物（重组蛋白）的分析技术
三、【实验步骤】
Pilot Expression
1. Inoculate 2 ml of SOB containing ampicillin (50 µg/ml) and chloramphenicol (35 µg/ml) with a single recombinant E. coli colony. Grow overnight at 37°C with shaking. 2. The next day, inoculate 25 ml of SOB (it is not necessary to include antibiotics for expression) to an OD600 of 0.1 with the overnight culture. (指导教师已 准备) 3. Grow the culture at 37°C with vigorous shaking to an OD600 = 0.4-0.6.
4. Remove a 1 ml aliquot of cells prior to IPTG induction, centrifuge the sample in a microcentrifuge, and aspirate the supernatant. Freeze the cell pellet at -20°C. This will be the time zero sample. 5. Add IPTG to a final concentration of 1 mM (0.25 ml of 100 mM IPTG stock to 25 ml culture) and continue to grow the cells. See page 12 for preparation of the IPTG stock solution. 6. After 1 hour of incubation, remove a 1 ml sample, centrifuge as described in Step 4, aspirate the supernatant, and freeze the cell pellet at -20°C. Continue to take samples at 1 hour intervals for 4 to 6 hours. （本次实验在 1h，3h取样）
The metal binding domain of the fusion peptide allows simple purification of recombinant proteins by Immobilized Metal Affinity Chromatography with Invitrogen’s ProBond. resin (available in bulk, see page v). The enterokinase cleavage recognition site in the fusion peptide located between the metal binding domain and the recombinant protein allows for subsequent removal of this N-terminal fusion peptide from thepurified recombinant protein.
Regulation of Expression of the Gene of Interest
Expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 (Φ10). T7 RNA polymerase specifically recognizes this promoter. For expression of the gene of interest, it is necessary to deliver T7 RNA polymerase to the cells by either inducing expression of the polymerase using the gratuitous inducer isopropyl β-Dthiogalactoside (IPTG), or infecting the cell with phage expressing the polymerase. Once sufficient T7 RNA polymerase is produced, it binds to the T7 promoter and transcribes the gene of interest.
E.coli
实验内容 1
观察pUC18-EGFP的表达，即观察该载体转化 细菌细胞后，表达目的蛋白-绿色荧光蛋白的 情况。收集细胞，在倒置荧光显微镜下观察， 细胞是否产生绿色荧光。
实验内容 2
含有目的基因LacZ的重组质粒pRSET-LacZ转 化细菌后的诱导表达分析。
启动子：原核启动子 (原核生物RNA 聚合酶不能识别真核基因的启动子)
Invitrogen
pRSET
ampicillin-resistant
克隆用菌株 Top10F’ 表达时采用菌株 BL21(DE3)pLysS
多克隆位点MCS
LacZ基因克隆入BamHI、HindIII位点
LacZ基因 3060bp 编码1019个氨基酸 产物为 半乳糖苷酶 该酶分解X-Gal （半乳糖苷类似物）呈现 蓝色
OD600 = 0.4-0.6
37℃ overnight
IPTG
诱导表达4-6小时，time-course expression
SDS-PAGE分析
Invitrogen
T7 promoter: bases 20-39 Ribosome binding site: bases 84-90 6xHis tag: bases 112-129 T7 gene 10 leader: bases 133-162 Anti-XpressTM epitope: bases 169-192 Multiple cloning site: bases 202-248 pRSET reverse priming site: bases 295-314 T7 transcription terminator: bases 256-385 f1 origin: bases 456-911 bla promoter: bases 943-1047 Ampicillin (bla) resistance gene (ORF): bases 1042-1902 pUC origin: bases 916-2852 (C)