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菌株保存

原核表达菌株的选择及菌种保存需知(Rosetta、BL21、Origami)

从生物通上看到一篇文章,介绍原核表达菌株的选择。文章很好,是生物通从Merck(Novagen隶属Merck旗下)的网站上编译而成的。这里摘取其中我认为重要的地方,并添加了一些EMD (Emanuel Merck Darmstadt)原网站上的内容。仅供参考。

菌株保存

在原核蛋白表达过程中,选择构建一个合适原核表达体系需要综合考虑3大因素:表达载体、宿主菌株、表达诱导条件,以获得最满意的表达效果。(如上图)

事实上,在平时的实验中,最容易被忽视的就是宿主菌的选择——多数人会直接选择自己实验室曾经用过的表达菌株,或者是载体配套的菌株,而不去追究原因——即使表达结果不佳,大多在表达条件和载体上找原因,也不会考究菌株的选择是否适合。

宿主细胞对原核表达可能会产生哪些影响呢?

菌株内源的蛋白酶过多,可能会造成外源表达产物的不稳定,所以一些蛋白酶缺陷型菌株往往成为理想的起始表达菌株。堪称经典的BL21系列就是lon和ompT 蛋白酶缺陷型,也是我们非常熟悉的表达菌株。大名鼎鼎的BL21(DE3)融源菌则是添加T7聚合酶基因,为T7表达系统而设计。

真核细胞偏爱的密码子和原核系统有不同,因此,在用原核系统表达真核基因的时候,真核基因中的一些密码子对于原核细胞来说可能是稀有密码子,从而导致表达效率和表达水平很低。改造基因是比较麻烦的做法,Rosetta 2系列就是更好的选择——这种携带pRARE2质粒的BL21衍生菌,补充大肠杆菌缺乏的七种(AUA, AGG, AGA, CUA, CCC, GGA 及CGG)稀有密码子对应的 tRNA,提高外源基因、尤其是真核基因在原核系统中的表达水平。(已经携带有氯霉素抗性质粒)

当要表达的蛋白质需要形成二硫键以形成正确的折叠时,可以选择K–12衍生菌Origami 2系列,thioredoxin reductase (trxB) 和glutathione reductase (gor)两条主要还原途径双突变菌株,显著提高细胞质中二硫键形成几率,促进蛋白可溶性及活性表达。(卡那霉素敏感)

Rosetta-gami 2则是综合上述两类菌株的优点,既补充7种稀有密码子,又能够促进二硫键的形成,帮助表达需要借助二硫键形成正确折叠构象的真核蛋白。(卡那霉素敏感)

Origami B是衍生自 lacZY突变的 BL21菌株,这个突变能根据IPTG的浓度精确调节表达产物,使得表达产物量呈现IPTG浓度依赖性。(四环素敏感)

菌株保存

在决定试用这些名字古怪的菌株时,有几个小Tips要注意的,一个是不同菌株有时已经携带某个质粒或者已经具有某种抗生素抗性,要注意自己的表达质粒是否能与之兼容。比如Rosetta 2 已经携带有氯霉素抗性质粒,不能再用氯霉素筛选等等。

原核表达常遇到的问题及推荐使用的菌株

菌株保存

重组质粒和菌株的保存建议

在实验室里保存重组菌株的时候,为了挑菌和传代方便,我们常用LB平板保存在4℃冰箱中,需要提质粒和表达接种的时候,就直接从平板上挑菌,但是这种方法对于重组质粒的稳定性不利,容易出现质粒丢失、表达水平不稳定、菌株退化乃至发生污染等情况。我们建议:

1、长期存放菌株和pET重组子应该存在甘油-70℃保种。但是要注意高浓度甘油(> 10%)会导致质粒不稳定。请尽量保持甘油浓度8%。(15%浓度的甘油保种液和新鲜菌液1:1就行)

2、重组质粒不宜长期保存于表达菌株(带DE3的大肠杆菌)中,请尽量使用带有recA endA 突变的克隆菌株(比如C600,DH5a)进行质粒抽提和保存质粒。表达型的菌株提质粒经常会有质量不高或者背景的问题。特别是大量抽提。

BL21、BL21(DE3)、BL21(DE3) pLysS三者基因型比较

首先介绍一下大肠杆菌常见的两个系列:

K-12系列:(DH5а属于K-12,DH5а的父本菌为DH1)

E. coli K-12 restricts DNA which is not protected by adenine methylat ion at sites AA*C[N6]GTGC or GCA*C[N6]GTT, encoded by the hsdRMS gene s(EcoKI). Deletions in these genes removes either the restriction or methylation or both of these functions.

B系列:(BL21属于B系列)

E. coli B derivative strains contain an hsdRMS system (EcoBI) restric ting and protectiing the sequence TGA*[N8]TGCT or AGCA*[N8]TCA.

基因型比较:

BL21:F- ompT gal dcm lon hsdS

(r B- m B-)

B

BL21(DE3):F- ompT gal dcm lon hsdS B(r B- m B-) λ(DE3)

BL21(DE3)pLysS:F- ompT gal dcm lon hsdS B(r B- m B-) λ(DE3) pLysS(cm R)

查看图示比较:http://www.wendangku.net/doc/d95a62f5f61fb7360b4c6598.html/user1/2081/archives/2008/124334.shtml

附说明:(大肠杆菌基因型及遗传符号说明)

DE3是整合在细菌基因组上的一种携带T7 RNA聚合酶基因和lacI基因的λ噬菌体,其基因型为:lacI lacUV5-T7 gene 1 ind1 sam7 nin5

pLysS= contains pLysS plasmid carrying chloramphenicol resistance and phage T7 lysozyme, effective at attenuating activity of T7 RNA polymerase, for better inhibition of expression unde r non-induced conditions. (pLysS 质粒上携带氯霉素抗性基因和T7 lysozyme基因,后者可以有效抑制T7 RNA聚合酶的水平,降低本底表达)

This web page was produced as an assigment for an undergraduate cours e at Davidson College.

The pET Expression System

Introduction

Expression systems are designed to produce many copies of a desired p rotein within a host cell. In order to accomplish this, an expression vector is inserted into a host cell. This vector contains all of the genetic coding necessary to produce the protein, including a promote r appropriate to the host cell, a sequence which terminates transcrip tion, and a sequence which codes for ribosome binding (Purves et al., 2001). One expression system was developed in 1986 by W. F. Studier and B. A. Moffatt, who created an RNA polymerase expression system wh ich was highly selective for bacteriophage T7 RNA polymerase. The ini tial system involved two different methods of maintaining T7 RNA poly merase into the cell - in one method, a lambda bacteriophage was used to insert the gene which codes for T7 RNA polymerase, and in the oth er, the gene for T7 RNA polymerase was inserted into the host chromos ome (Studier et al, 1986). This expression system has become known as the pET Expression System, and is now widely used because of its abi lity to mass-produce proteins, the specificity involved in the T7 pro moter which only binds T7 RNA polymerase, and also the design of the system which allows for the easy manipulation of how much of the desi red protein is expressed and when that expression occurs. (Unger, 199 7; Novagen, 2002-2003).

Producing Proteins with the pET Expression System

Designing the pET Plasmid:

A pET vector is a bacterial plasmid designed to enable the quick prod uction of a large quantity of any desired protein when activated. Thi s plasmid (pictured below) contains several important elements - a la cI gene which codes for the lac repressor protein, a T7 promoter whic h is specific to only T7 RNA polymerase (not bacterial RNA polymerase) and also does not occur anywhere in the prokaryotic genome, a lac op erator which can block transcription, a polylinker, an f1 origin of r eplication (so that a single-stranded plasmid can be produced when co -infected with M13 helper phage), an ampicillin resistance gene, and a ColE1 origin of replication (Blaber, 1998).

To start the process, your favorite gene (YFG) is cloned into a pET p lasmid at the polylinker site. Both the T7 promoter and the lac opera tor are located 5' to YFG. When the T7 RNA polymerase is present and the lac operator is not repressed, the transcription of YFG proceeds rapidly. Because T7 is a viral promoter, it transcribes rapidly and p rofusely for as long as the T7 RNA polymerase is present (Campbell, 2 003). The expression of your favorite protein (YFP) increases rapidly

as the amount of mRNA transcribed from YFG increases. Within a few h ours, YFP is one of the most prevalent components of the cell. (Unger, 1997).

菌株保存

Figure 1: The pET Vector. This plasmid contains a drug resistant marker for ampicil lin resistance (green), the lacI gene (blue), the T7 transcription promoter (red), the lac operator region (pale green) 3' to the T7 promoter, and a polylinker region (black). Also, there are two origins of replication - one is the f1 origin which e nables the production of a single stranded vector under appropriate conditions, and the other is the conventional origin of replication. This image was used with perm ission from Dr. Michael Blaber, of Florida State University. The original reference can be found here.

Introducing T7 RNA Polymerase to the Host Cell:

One of the most important parts of the pET Expression System involves the fact that YFG is not transcribed unless the T7 RNA polymerase is present. Prokaryotic cells do not produce this type of RNA, and ther efore the T7 RNA polymerase must be added. Usually, the host cell for this expression system is a bacteria which has been genetically engi neered to incorporate the gene for T7 RNA polymerase, the lac promote r and the lac operator in it's genome. When lactose or a molecule sim

ilar to lactose is present inside the cell, transcription of the T7 R NA polymerase is activated.. Typically, the host cell used is E. coli strain BL(DE3) (Blaber, 1998). The diagram below shows the genome of the host cell.

菌株保存

Figure 2: The Host Chromosome. The lac promoter is in red, the lac operator is gree n, the gene which encodes the T7 RNA polymerase is pink, and the lac inducer is blu e. The host cell genome is represented by the black line, and the brown line repres ents the cell membrane. This image was used with permission from Dr. Michael Blaber, of Florida State University. The original reference can be found here.

T7 RNA polymerase can be introduced to the cell through methods other than incorporation a gene in the host chromosome. It can be introduc ed through infection of the original host cell with lambda CE6 (Novag en 2003-2003).

Controlling the pET Expression System

Control of the pET expression system is accomplished through the lac promoter and operator. Before YFG can be transcribed, T7 polymerase m ust be present. The gene on the host cell chromosome usually has an i nducible promoter which is activated by IPTG. This molecule, IPTG, di splaces the repressor from the lac operator. Since there are lac oper

ators on both the gene encoding T7 polymerase and YFG, IPTG activates both genes (Science Advisory Board, 2003; Campbell, 2003). Therefore, when IPTG is added to the cell, the T7 polymerase is expressed, and quickly begins to transcribe YFG which is then translated as YFP.

IPTG works to displace a lac repressor since IPTG is an analogue of l actose (Blaber, 1998). The lac genes express enzymes which are involv ed in the breaking down of lactose, and therefore, the presence of la ctose (or it's analogue) would trigger the initiation of transcriptio n of lac genes.

References

Blaber M. 1998 Spring. Web Page for Lecture 25 of Molecular Biology a nd Biotechnology Course: Prokaryotic Expression Vectors. . Accessed 2003 Feb17.

Campbell M.. 2003 Jan 31. Class Lecture.

Heller HC, Orians GH, Purves WK, Sadava D. 2001. Life: The Science of Biology. 6th ed. Sunderland (MA): Sinauer Associates, Inc.; p 251, p 322-323

Novagen.. 2002-2003. Protein Expression: Prokaryotic Expression: pETB lue and pET System Overview. Novagen 2002-2003 Catalog. p 84-91. . Accessed 200 3 Feb 17.

Novagen. 2001. Protein Expression: Prokaryotic Expression: pET System Overview. 2001 Novagen Catalog. p 68-72. . Accessed 2003 Feb 17.