当前位置:首页 >> >>

Protein


Protein

Expression of Proteins in E. coli
Expression of a recombinant protein can be approached in general by constructing a plasmid that encodes the desired protein, introducing the plasmid into the required host cell, growing the host cells and inducing protein expression, and then lysing the cells, purifying the protein, and performing SDS-PAGE analysis to verify the presence of the protein (Figure 1). The protocols and recommendations given in the Plasmid DNA chapter for the handling and transformation of E. coli are also valid for the production of recombinant proteins. With careful choice of host strains, vectors, and growth conditions, most recombinant proteins can be cloned and expressed at high levels in E. coli. Optimal growth and expression conditions for the protein of interest should be established with smallscale cultures before large-scale protein purification is attempted. Generation of Recombinant Proteins
Plasmid

neo

Transformation of bacteria
lacI

Bacterial growth

Protein purification

Protein analysis

Figure 1. Overview of the steps involved in expression and analysis of recombinant proteins.

Basic principles
This section discusses critical factors to be considered when expressing foreign proteins in E. coli. Culture media The media of choice for the growth of E. coli cells containing an expression plasmid are LB medium and its modifications, 2x YT, or Super Broth, each containing the relevant selective antibiotic(s). Initially it is advisable to try expression in all three media in parallel, and to do a time course analysis to monitor growth and expression after induction. Striking differences between the level of expression in different media and at different times are often observed. Maintenance of the expression plasmid Poor plasmid maintenance in the cells can lead to low expression levels. Ampicillin is an unstable antibiotic and is rapidly depleted in growing cultures due in part to the β-lactamase secreted by resistant bacterial cells. It is important to check plasmid levels by plating cells from the expression culture on plates with and without ampicillin. If the stability of the expression construct is a problem, the cultures should be grown in the presence of 200 ?g/ml ampicillin, and the level should be maintained by supplementing ampicillin during long growth periods. Alternatively, the cultures may be grown in the presence of carbenicillin, a more stable β-lactam, at 50 ?g/ml (see “Antibiotics”, page 5).

Protein

70

Protein

Small-scale expression cultures Small-scale expression and purification experiments are highly recommended and should be performed before proceeding with a large-scale preparation. In many cases aliquots of the cells can be lysed in a small volume of sample buffer and analyzed directly by SDS-PAGE. The use of small expression cultures provides a rapid way to judge the effects of varied growth conditions on expression levels and solubility of recombinant proteins. Expression levels vary between different colonies of freshly transformed cells, and small-scale preparations permit the selection of clones displaying optimal expression rates. Induction of protein expression The method used for induction of protein expression is dependent on the plasmid vector and E. coli strain used. Protein expression can be induced by a raising of the incubation temperature or by the addition of an inducing chemical such as isopropyl-β-D-thiogalactoside (IPTG) to the culture medium. Details of induction methods and the plasmids they relate to can be found in standard molecular biology texts (1,2). Time-course analysis of protein expression To optimize the expression of a given protein construct, a time-course analysis by SDS-PAGE (Protocol 5, page 75) of the level of protein expression is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the protein present at various times after induction, the optimal induction period can be established (Figure 2). Time-Course Analysis of Protein Expression
M C 0.5 h 1h 2h 3h 4h

?

Figure 2. Time course analysis of the expression of dihydrofolate reductase (arrowed). Aliquots were removed at the times indicated and analysed by SDS-PAGE. C: uninduced control. M: markers.

Colony blots
We recommend the colony-blot procedure (Figure 3; Protocol 1, page 72) to identify clones expressing a protein and to distinguish semi-quantitatively between expression rates. This can be an advantage for selecting clones after transformation, since freshly transformed colonies may differ significantly in their expression rates. Using this method, colonies subsequently found to be expressing proteins at rates as low as 0.1 to 0.5 mg/liter are easily distinguished from colonies that do not express protein. Note: If using the QIAexpress? Expression System, the small size of the His tag means that small peptides (<30 amino acids) expressed from QIAexpress vectors without an insert are degraded within the cells, and will not yield a false positive signal in the detection procedure. Other commonly used vectors that encode larger affinity tags may lead to expression of a small, but stable and detectable translation product even without an insert. This will lead to false positive signals from colonies that harbor the expression vector without insert, which may be indistinguishable from the signals from colonies expressing the desired protein. Colony-Blot Procedure
Make replica with nitrocellulose membrane Transfer nitrocellulose membrane to new plate and induce expression

Master plate (original transformants) – IPTG + IPTG (4 h)

Pick positive clones from master plate

Membrane with positive signals after alkaline lysis and western blot procedure

Figure 3. Detection of positive expression clones by colony blotting.

Protein

71


赞助商链接
相关文章:
Protein_A亲和介质说明书
产品简介 Protein A 能特异性地与抗体的 Fc 区结合, 所以 Protein A 亲和介质可用于抗体 (单 抗和多抗)的分离纯化,经过一步亲和层析,即可从腹水、血清和...
Protein A Resin纯化抗血清
Protein A Resin纯化抗血清_生物学_自然科学_专业资料。Protein A Resin(Cat.No.L00210)纯化抗血清操作程序 1 实验原理 Protein A 亲和层析介质是纯化和分离 ...
E-D2A XXXX产品Protein A含量测定标准操作程序0507 4
文件名称:XXXX 产品 Protein A 含量测定标准操作程序 文件编号: E-D1A 起草人/日期: 生效日期: 版本号:01 审核人/日期: 页码: 1 / 5 批准人/日期: 分发...
补充蛋白质与运动能力
例如,奶中的乳清蛋白 (WheyProtein)、 蛋类中的卵清蛋白。 现在流行的多种蛋白类补充食品, 如牛奶、 鸡蛋蛋白、 大豆蛋白、牛肉甚至蔬菜蛋白等,牛奶中大约含 ...
蛋白质在人体中的功能与作用
蛋白质的英文是 protein,源于希腊文的 proteios,是“头等重要”意思,表明蛋白质是生 命活动中头等重要物质。蛋白质是细胞组分中含量最为丰富、功能最多的高分子...
Protein A, ProteinG
蛋白量约为 200 微克至 1 毫克,加入约 1 微克和免疫 沉淀时使用的 IgG 种属相同的普通 IgG 和 20 微升充分重悬的 Protein A+G Agarose,4℃缓 慢摇动 30...
protein A与proteinG的区别
protein A与proteinG的区别_理学_高等教育_教育专区。分享一下,自己所找的一些关于IP的实验资料,希望对大家有用!!中文名称 重组纯化蛋白 A 英文名称 Recomb ...
Protein A 、Protein G 与抗体的结合
Protein A 、Protein G 与抗体的结合 1 Protein A Sepharose 、rProtein A Sepharose 亲和层析介质与抗体的结合 目前,约 70-80%的抗体纯化使用 Protein A、...
Protein G Beads使用说明书
Protein G Beads 使用说明书【产品介绍】 产品介绍】 Protein G Beads 是将重组 Protein G 蛋白分子共价交联到 4%琼脂糖胶粒上制成。 本产品使用特殊试剂对琼脂...
Protein G亲和介质说明书
Protein G亲和介质说明书_医药卫生_专业资料。国家生化工程技术研究中心(北京) Protein A 亲和介质(Protein G QZT 4FF)产品说明书 亲和介质( ) 1. 产品简介...
更多相关标签: