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Expression of protein in prokaryotic cells


Expression of Protein in Prokaryotic Cell

北京大学医学部 基础医学院 细胞生物学系 甄红英

Expression of the fluorescent GFP-CBP fusion protein in bacterial (Panel A) and mammalian cells (Panel B) is shown

The Western blot in Panel C Lane 1: CHO cell lysates. Lane 2: Bacterial cell lysates. Lane 3: Commercially available recombinant GFP for size comparison.

主要过程
制备pET 载体 制备

制备插入DNA 制备插入 将插入的DNA片断克隆到 片断克隆到pET 载体 将插入的 片断克隆到

转化表达质粒到宿主菌 诱导/优化表达目的蛋白 诱导 优化表达目的蛋白

放大实验

纯化目的蛋白

Procedure of Recombinant Protein in Vitro Target gene Choice of vector Ligation Transformation Screen and identify positive clones Sequence Expression and purification target protein

SourceSource of target gene of target gene Genomic DNA cDNA Chemical synthesis PCR amplification

表达载体的选择
蛋白质
融合蛋白 天然蛋白

基因
原核基因 真核基因

可分泌性 融合蛋白的加工 启动子

pET载体系统 载体系统
目的基因被克隆到pET质粒载体 质粒载体 目的基因被克隆到 受噬菌体T7强转录及翻译 可选择) 强转录及翻译( 受噬菌体 强转录及翻译(可选择)信号控制 表达由宿主细胞提供的T7RNA聚合酶诱导 表达由宿主细胞提供的 聚合酶诱导 两种启动目的蛋白表达的方式 带有受λ 和 启动子控制的 启动子控制的T7RNA聚合酶的 聚合酶的λ 带有受 pL和pI启动子控制的 聚合酶的 CE6噬菌体侵染宿主细胞 噬菌体侵染宿主细胞 将质粒转入带有受lacUV5控制的 控制的T7RNA聚合酶 将质粒转入带有受 控制的 聚合酶 基因的表达型细胞

选择合适的pET载体 载体 选择合适的
转录载体 表达本身带有原核核糖体结合位点和AUG起 表达本身带有原核核糖体结合位点和 起 始密码子的目的基因 3种:pET-21(+)、 pET-24(+)和 pET-23(+) 种 、 和 翻译载体 来自T7噬菌体主要衣壳蛋白的高效核糖体结合位点 来自 噬菌体主要衣壳蛋白的高效核糖体结合位点 用于表达那些不带有核糖体结合位点的目的基因

选择合适的pET载体 载体 选择合适的
翻译载体在命名上与转录载体不同, 翻译载体在命名上与转录载体不同,多一个字母后缀 pET-21a(+) 表示相对于BamHI克隆位点识别序列 表示相对于 克隆位点识别序列GGATCC的阅 的阅 克隆位点识别序列 读框,所有带后缀a的载体从 的载体从GGA三联密码子开始表达; 三联密码子开始表达; 读框,所有带后缀 的载体从 三联密码子开始表达 pET-21b 的从GAT开始; 开始; 带b的从 的从 开始 pET-21c 带c的从 的从BamHI识别序列 识别序列ATC三联密码子开始; 三联密码子开始; 的从 识别序列 三联密码子开始 pET-21d 后缀的载体阅读框和带c的一样 带d后缀的载体阅读框和带 的一样,不同的是它们有 后缀的载体阅读框和带 的一样, 一个上游Nco I克隆位点而非 克隆位点而非Nde I位点以便直接将目的基 一个上游 克隆位点而非 位点以便直接将目的基 因克隆到AUG起始密码子 因克隆到 起始密码子

选择合适的pET载体 载体 选择合适的
基本考虑因素 1. 目的蛋白的应用 2. 目的蛋白已知特定信息 3. 克隆策略 细胞溶解性及细胞定位 载体可以通过三种方式改善目的蛋白的溶解性或正确折叠: 载体可以通过三种方式改善目的蛋白的溶解性或正确折叠: (1) 与本身溶解性高的多肽序列融合表达 例如谷胱甘肽 转移 与本身溶解性高的多肽序列融合表达[例如谷胱甘肽 例如谷胱甘肽-S-转移 硫氧还蛋白(Trx)及NusA (N utilization substance A)] 酶(GST), 硫氧还蛋白 及 (2) 与催化二硫键形成的酶融合表达 例如 与催化二硫键形成的酶融合表达[例如 例如Trx, DsbA, 及DsbC] (3) 与信号序列融合表达,输出到细胞周质。如采用蛋白定位与 与信号序列融合表达,输出到细胞周质。 细胞质的表达载体, 细胞质的表达载体,可选用允许二硫键在胞质中形成的宿主菌 株来使目的蛋白正确折叠 包涵体有利于纯化: )离心收获浓度高; ) 包涵体有利于纯化:1)离心收获浓度高;2)免受蛋白酶水解

Removal of Tags
Thrombin Factor Xa Leu Val Pro Arg Gly Ser Ile Glu Gly Arg

Enterokinase Asp Asp Asp Asp Lys

pET 系统蛋白表达的调控

pET Blue and pET System

TM

pET System Features
The number-one cited system for prokarytic protein expression Lowest basal expression levels of any E.coli expression system “Tunable” control for modulating expression levels is desired Wildest variety of fusion tags and configuration of any expression system Specialized vectors and hosts for production of soluble proteins,disulfide bond formation, protein export and peptide production, ect.

pET Blue System
TM

pET Blue System Feature
Blue/white screening for easy clone High-copy number for high plasmid-DNA yields Available as AcceptorTM vector or perfectly Blunt for rapid PCR cloning No basal expression for target gene; eliminates plasmid instability associated with toxic gene products Same expression levels as classic pET vectors “Tunable” control of expression levels with tunerTM (DE3) pLacI and RossetaTM (DE3) pLacI host strains

TM

Using vectors to solve problem
Poor expression Leaky expression Unstable expression Difficult purification Insoluble protein Need for unfused protein

Purification and detection tags
Tag His-Tag sequence GST-Tag sequence T7-Tag sequence S-Tag sequence N/C terminal Size(aa) Application Western blot N or C 6,8or10 Purification N 220 Western blot Purification Quantitative assay Western blot Immunoprecipitation Purification Western blot Purification Quantitative assay

N N

11 15

Using hosts to solve problems Proteolysis Recombination Leaky expression Poor expression Truncated protein Misfolded protein

Transformation Calcium Chloride Electroporation

Recovering the target protein
Verify target protein Lyse cells Remove nucleic acids Opimize purification Remove tags Transfer to storage buffer

主要步骤 制备pET 载体 制备





1.用限制性酶消化 去磷酸化 , 或使用 easy载体 用限制性酶消化, 或使用T载体
2.琼脂糖电泳纯化载体 琼脂糖电泳纯化载体DNA(或使用 easy载体) 或使用T载体) 琼脂糖电泳纯化载体 或使用 载体 1. 纯化 纯化PCR 2. 限制性酶消化 3.琼脂糖电泳纯化 琼脂糖电泳纯化DNA 琼脂糖电泳纯化 1. 插入片断与 插入片断与pET 载体连接 2. 转化非表达型宿主菌 3. 筛选阳性克隆;菌落 筛选阳性克隆;菌落PCR,制备质粒 ,制备质粒DNA 通过测序确定阅读框, 通过测序确定阅读框,或进行体外转录翻译 1.转化带有 转化带有T7RNA聚合酶基因的菌株或(λDE3 聚合酶基因的菌株或( 转化带有 聚合酶基因的菌株或 溶原菌) 感染非DE3宿主菌 溶原菌)以λ CE6感染非 感染非 宿主菌 1. 检测质粒稳定性(选择性操作) 检测质粒稳定性(选择性操作) 2. 确定细胞及亚细胞组分,表达时间和温度条 确定细胞及亚细胞组分, 件; 分析蛋白溶解性及活性 3.用SDS-PAGE, Western Blot, 定量分析,确定 定量分析, 用 目的 蛋白 1. 放大培养 2. 制备粗提物 3. 亲和纯化 4. 切去融合标签并去除蛋白酶(如果必要) 切去融合标签并去除蛋白酶(如果必要)

制备插入DNA片断 制备插入 片断 将插入DNA片断克隆到 片断克隆到pET 载体 将插入 片断克隆到

转化表达质粒到宿主菌 菌 诱导/优化表达目的蛋白 诱导 优化表达目的蛋白

放大实验 纯化目的蛋白

QIAGEN PCR Cloning System

Protein expression checklist
Promoter Source of RNA polymerase Ribosome binding site Readsing frame Drug selection Storage of construct Medium Induction condition Detection method Storage of protein

IMPACT -TWINS

TM

pTWINS

IMPACT -TYB11

TM

pTYB11


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