Chapter 23

recombinant DNA technology

(genetic engineering)

授课老师：陈蔚文

山东大学医学院生物化学与分子生物学系

SetcionⅠ

DNA Recombination and Gene Transfer

Occur Frequently in Nature

DNA recombination

conjugation

transformation

transduction

transposition

homologous recombination

site-specific recombination

SetcionⅡ

DNA Recombination Technique

DNA Cloning

Molecular Cloning

Genetic engineering

5

Naissance of gene engineering

Theory basis:

•1865, G.J.Mendel

•1944, O.T. Avery

•1953, double helix

•1961, control of gene expression

Technology basis:

•Small molecular vector: plasmid, virus, phage

•Restriction endonuclease: cut DNA in vitro

•DNA ligase: ligate DNA in vitro

6

DNA Stores Genetic Information

replication replication

transcription transcription

translationtranslation

Recombinant DNA Technology

8

History

•1972, Boyer, EcoRⅠ

•1972, P. Berg, SV40 & λ phage were cut with EcoRⅠ,

then ligated with T4 DNA ligase — first recombinant

experiment in vitro, 1980 Nobel prize

•1973, Cohen, R6-5 (Kan+) & pSC101 (tet+) were cut

with EcoRⅠ and then ligated with T4 DNA ligase,

resisted kan and tet.

9

1974, Cohen & Boyer, a gene from Xenopus laevis was recombined with pSC101 and transformed into E. coli, the mRNA of this gene was detected into E. coli —first gene clone experiment

1974, P. Berg , NIH, Recombinant DNA Advisory Committee

1976, NIH, rule for Recombinant DNA study

1977, Boyer founded Genentech with venture capitalist Robert A. Swanson.

1997, Cloned sheep Dolly

Concept： DNA cloning

Materials:

– Enzymes

– Target DNA

– Vectors

– Host cells

Basic strategy

Content：

Clone

A population of identical cells or DNA

molecules descended from a single

progenitor. Viruses or organisms that

are genetically identical and

descended from a single progenitor.

In vitro, attach the target DNA fragments to the

genetic elements that can autonomously

replicating (carrier DNA) , to form recombinant

DNA molecules, and then introduced into

recipient cell to replicate and amplify, resulting

in a large number copies of a single DNA

molecule.

DNA Cloning

Definition:

Recombinant DNA/Gene cloning /DNA cloning

With some methods or

techniques recombinant /

chimeric DNA molecules are

formed by joining different

DNA fragments from different

sources in vitro.

The recombinant DNA molecules

are introduced into appropriate

host cells in which they can be

replicated or expressed. The host

cells can reproduce and all of its

descendants carrying the same

recombinant DNA molecules are

called a clone. So the

recombinant DNA is cloned by

this process.

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of

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ing

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Materials and tools for DNA cloning include:

Target DNA: DNA fragment or gene you want

to study

Vehicle: Plasmids or viral vectors

Scissors: Restriction enzymes

Glue: DNA ligase

Host cells:prokaryotes or eukaryotes

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Ⅰ Tool enzyme

1. Restriction Endonucleases

Restriction endonuclease : Site-specific endodeoxyribonucleases that

cleave both strands of DNA at points in or near

the specific site recognized by the enzyme;

important tools in genetic engineering.

Restriction-modification system

in bacteria

restriction endonuclease and methylase

restriction endonuclease can cleave foreign DNA;

“restrict” the entry of DNA, protect bacteria own

DNA

3681 kinds restriction endonuclease, type I, II and III

22

Restriction endonuclease Ⅱ

Cut DNA sequence-specifically;

Named (e.g., EcoRI) for bacterial genus,

species, strain, and type;

Recognize specific 4~8 bp sequences

that have symmetry (palindrome);

The cut ends are blunt or sticky/

cohesive ends;

A key tool in recombinant DNA research.

Restriction-modification system

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The interaction of EcoRI endonuclease with its target sequence.

RE

Phosphodiester bond

Restriction enzymes are named after the

bacterium from which they are isolated.

eg. EcoR I

The first letter

of the genus

the first two letters

of the species

The first letter

of the strain

The order of

discovery

The endonucleases recognize and cleave a specific double-stranded DNA sequence that is 4~8bp long sequence.

sticky end

blunt end

The DNA cuts produced by restriction enzymes have two types of ends depending on the cut position by the enzymes.

EcoRI

PstI5'

3'

3'

-G ACGTC-

-CTGCA G-

5'

5' 3'-G AATTC-

5'3' -CTTAA G-

3' sticky end

5' sticky end

Sticky ends are particular useful in constructing

recombinant or chimeric DNA molecules.

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Using the same enzyme to cut DNA molecules from different sources is most useful in recombinant DNA technology.

intermolecular ligation

GGATCC

CCTAGG

G

CCTAG

GATCC

G

BamHI

GGATCCCCTAGG

GCCTAG

GATCCG+

Bam HⅠ

GGATCCCCTAGG

GCCTAG

GATCCG+

BstⅠ

Isoschizomer are pairs of restriction enzymes specific to the same recognition sequence, isolated from different strains of bacteria and therefore may require different reaction conditions

BamH Ⅰ

Sau3A Ⅰ

5' 3'-G GATCC-

5'3' -CCTAG G-

5'

5'3'

3'-N GATCN-

-NCTAG N-

Compatible end

Isocaudomers are pairs of restriction enzymes

that have slightly different recognition sequences

but upon cleavage generate identical termini

2.DNA Ligase: another important and useful

enzyme in recombinant DNA technology.

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DNA ligase can join together any two DNA fragments

in vitro to produce recombinant DNA molecules.

The insertion of a DNA fragment into a bacterial

plasmid with the enzyme DNA ligase

nick

gap

Nick

Ⅱ Target DNA interest

Genomic DNA

cDNA

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Ⅲ Vector DNA

42

Vector DNA is a DNA molecule capable of self-replication

in a host organism and into which a piece of

DNA can be inserted, including cloning vector

and expressing vector.

Self-replication in host cells: the origin for

replication is required for the vector to

propagate.

Several unique recognition sequences for

different restriction enzymes that can provide

sites where the vector can be cut and foreign

DNA fragment can be inserted.

Marks for selection of the cells that contain the

vector or recombinant vector

1. Cloning vector

① Cloning vectors must have 3 basic features

The constructed

E. coli plasmid

pBR322.

insert

replication

BamHI

②Plasmids:

small, circular, duplex DNA molecule ususally1~200kb size.

exist in the cytosol of the bacterium, symbiotic with the host.

replicate independently of the bacterial DNA.

confer antibiotic resistance to the host cell.

many constructed plasmids are available commercially.

their complete sequences are known.

some nonessential sequences are removed.

synthetic polylinker with unique sites forseveral restriction enzymes is inserted.

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48

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Some of the important features of pBR322

Several unique recognition sequences for different restriction endonucleases provide sites where the plasmid can be cut and foreign DNA inserted.

Have a origin of replication ( ori )

Ampicilin resistance gene (ampr )、tetracycline resistance gene ( tetr ) allow the selection of cells

An overall small size which can facilitate the plasmid entry into cells.

Multiple cloning

site (MCS)

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1.Lac operon structure

regulatory region

coding region

Z:β-galactosidase

Y: permease

A: transacetylase

promoter

operator

lac operon

CAP binding site

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α-complementation

Some plasmid vectors such as pUC19 carry the lac Z’ gene that express α fragment of β-galactosidase. The α fragment is the N-terminus of the β-galactosidase. Typically, the mutant E. coli host strain only carry the ω fragment, which is the C-terminus of the protein. Either ω omega or α fragment alone is nonfunctional. When the vector containing lac Z’ introduced into mutant E. coli, both the α and ω fragments are present there is an interaction and a functionally intact beta-galactosidase protein can be produced. This interaction is called α complementation

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There are two categories of plasmids

Stringent plasmids：replicate only

when the chromosome replicates. 1~2

Relaxed plasmids：replicate on their

own. This gives you a higher ratio of

plasmids to chromosome. ≥10

Other cloning vectors

Lambda phage: λgt & EMBL

M13 phage: M13mp, pUC

Cosmid (cos site-carrying plasmid )

BAC (Bacterial Artificial Chromosomes )

YAC (Yeast artificial chromosome)

Vector Host Characteristics Insert size range

Plasmid bacteria, yeast Small circular DNA <5 - 10 kb

phage λ bacteria Linear viral DNA up to ~20 kb

Cosmid bacteria Hybrid of plasmid and phage up to ~50

BAC bacteria Small circular DN 300kb

YAC yeast DNA containing yeast centromere,

telomeresand origins of replication

~200 to ~1000 kb,

<2Mb

Vectors used in molecular cloning

Bacterial artificial

chromosomes (BACs) as

cloning vectors.

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Cloning Vectors must contain:

Origin of replication: DNA polymerase

Selectable marker(s): antibiotic resistance

Multiple cloning sites (restriction enzyme

sites): cutting/pasting of DNA fragments

2. Expression vector: Cloning vectors with the transcription and translation signals

Promoter: recognized by RNA polymerase

Production of large

amounts of a protein

from a protein coding

DNA sequence cloned

into an expression

vector and introduced

into cells.

①D

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Regulated expression

of RecA protein in a

bacterial cell.

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② Eukaryotic expression vector

Shuttle vector

Eukaryotic expression regulatory

elements

Yeast, insect, and mammal

Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms

ⅣHost cells:

1.easy to take up the recombinant DNA.

2.no selective markers that the vector contained.

3.no restriction for the replication of the

recombinant DNA.

4.no restriction enzymes that can cut the

recombinant DNA.

5.they are prokaryotes or eukaryotes

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ⅤCloning strategy

Separation: A specific gene or DNA fragment and

vector DNA you chosed are separated from their originals

Cut: Vector DNA and target DNA are cut at precise

locations by proper restriction enzymes.

Ligation: The target DNA is linked to the vector DNA

covalently by DNA ligase to form a recombinant DNA.

Transformation: The recombinant DNA is introduced

into a host cell that can provide the enzymatic machinery

for DNA replication.

Selection or screening: the host cells that contain the

recombinant DNA are identified by using some methods.

Cloning gene expression: proteins expression

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The procedures of gene cloning

Separation

Separating a specific gene or DNA fragment from its original as

target gene that you studied or you are interested in.

Acquirement of target gene:

(1). Separated directly from chromosomal DNA, easily in

prokaryotes.

digestion with restriction enzyme

separated by electrophoresis_

+

markers

samples

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(2) Artificial synthesis of simple and

short sequence.

working backward from the amino

acid sequence, deducing the DNA

sequence that would code for it

obtain the necessary DNA sequence

information from sequence databases

(3) Selection from gene library

Gene library: a collection of DNA clones, gathered

together as a source of DNA for sequencing, gene

discovery, or gene function studies. Include

genomic DNA library and cDNA library.

Genomic DNA library: A DNA library containing

DNA segments that represent all (or most) of

the sequences in an organism’s genome.

cDNA library: DNA library consisting entirely of

cloned cDNAs from a particular organism or cell

type under certain conditions.

80

The vector for construction of library

λ Phage

Cosmid

BAC: Bacterial artificial chromosome

YAC: Yeast artificial chromosome

*Phage λ DNA

- a bacteriophage that can

infect E.coli.

-linear double-stands DNA,

48502 bp in length.

-self-replication in host cell

The genome of λ phage consist of left arm, central portion and right arm. Both arms are required by lytic growth, but central portion is not essential for replication of λ DNA and can be broken or removed.

central portion (~15 kb)

Insertion /replacement vectors

DNA is packaged into infectious phage particles

only if it is between 40~53 kb long

Insertion vectors:

Foreign DNA sequence is inserted into

the λ genome without any significant change

of the wild type genome.

Smaller insert size (up to ~10kb).

They may contain a multiple cloning site

inserted in lacZ system for screening of

recombinant bacterial colonies.

Can be used to clone smaller DNA molecule.

Such as λ ZAP, λ gt, etc.

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Replacement vectors:

Full length λ molecule having two identical

restriction sites flanked by “stuffer fragment”.

Stuffer fragment is replaced by foreign DNAduring restriction cloning.

The vector without the foreign insert cannot bepackaged due to the size limitation (smallerthan the required).

Insert size ranges between 10-23 kb.

Eg. λ EMBL 3, λ EMBL 4, λ DASH etc.

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Genomic Library

a collection of bacteria (or bacteriophage) that contain all the DNA in the organism’s genome on plasmids; every gene in the entire genome is present in the genomic library

BamH Ⅰ 5' 3'-G GATCC-

5'3' -CCTAG G-

Sau3A Ⅰ5'

5'3'

3'-N GATCN-

-NCTAG N-

Replacement vector

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cDNA library

90

In construction of cDNA libraries we make use of

the fact that all translated mRNAs in eukaryotic

cells contain poly (A) tail.

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The

synth

esis

of

cDN

A.

94

95

cDNAs can

be introduced

into a

cloning /

expression

vector and

cloned in a

bacterial host

cDNA

The

dif

fere

nce

s b

etw

een c

DN

A c

lones

and g

eno

mic

DN

A c

lones

der

ived

fro

m

the

sam

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gio

n o

f D

NA

Clone everything you can and then find what you need.

How to clone a specific gene from gene library?

How to find the clone with "our" gene?

The most common methods include:

1. Phenotypic screening

2. DNA hybridization

3. Screening with antibodies

Phenotypic screening is used when cloned gene is

expressed and changes properties of the cell in an

"obvious way". In the shown example, the protein

encoded in the cloned gene changes the color of

transformed cells.

DNA hybridization

Use o

f hybridiz

ation to identify

a c

lone w

ith a

part

icula

r D

NA

segm

ent

Screening with antibodies is used when cloned gene is

expressed and antibodies recognizing the encoded

protein are available.

(4). Amplified by polymerase chain reaction(PCR) method.

DNA polymerase DNA

template

primerprimer

Amplified

DNA

fragment

108

(5) Other methods

yeast one-hybrid

yeast two-hybrid

Yeast two-hybrid analysis

Cut

Cutting vector DNA and target DNA at precise

locations by proper restriction enzymes.

Choice of the restriction enzymes:

The target gene has two sites for one enzyme

in its two flanks.

The vector has one or two sites for the

enzyme at proper location or MCS

Using the

same one

enzyme to

cut target

gene and

vector

respectively

113

Choice of the restriction enzymes:

The target gene has two sites for

two different enzymes in its two

flanks respectively.

The vector has the same two sites

for the two enzymes in MCS

Using the

same two

enzymes to cut

the target

gene and

vector DNA

respectively

xxxGAATTCxxxxxxxxxxxAAGCTTxxx

xxxCTTAAGxxxxxxxxxxxTTCGAAxxx

EcoRI Hind III

AATTCxxxxxxxxxxxA

GxxxxxxxxxxxTTCGA

EcoRI

HindIII

G AGCTT

CTTAA A

Choice of the restriction enzymes:

The target gene has more than two

sites for one enzyme not only in the

two flanks but also within the gene.

The vector has the same site for the

enzyme in MCS

Using the

same one

enzyme to cut

the vector and

to digest the

target DNA

partiallyPstI PstI PstI

marker

sampleExcise the

interest band

and purify it

Ligate the gene

with the vector

that is cut by PstI

- +

Choice of the restriction enzymes:

Sometimes in the target gene there are no suitable

sites that also exist in the vector for some restriction

enzymes. How to deal with this problem.

Using the different enzymes that are suitable to cut out

the target gene from big DNA molecule

modifying the ends with some enzyme to form blunt

ends adding a linker with the site that also

exist in the vector to the modified blunt ends

best choice: PCR

Joining the target DNA

to the vector DNA

covalently using DNA

ligase to form a

recombinant DNA

Ligation

There are 3 kinds of ends ligated:

1. Ligation between compatible sticky ends

2. Ligation between blunt ends

3. Ligation between sticky and blunt ends

Ligase

AATTCxxxxxxA

GxxxxxxTTCGAG AGCTT

CTTAA A

GAATTCxxxxxxAAGCTT

CTTAAGxxxxxxTTCGAA

ligase

*Using the same one enzyme to cut

*Ligated easily and conveniently

*Self-reconnection of vector

*Inserted in two directions

Sticky-end ligation

*Using the same two enzymes to cut

*Ligated easily and conveniently

*no self-reconnection of vector

*Inserted in one direction

EcoRI HindIII

EcoRI HindIIIEcoRI EcoRI

Ligation between sticky ends with synthetic linker

GAATTCGG

CTTAAGCC

GGGAATTC

CCCTTAAG

EcoRI

Cut with RE

Ligase + linker

*Cut target DNA using proper RE

*Ligating blunt-end with a linker

*Cut the vector and DNA with a linker

using the same restriction enzyme and

ligate them.

Ligation between homopolymer tails

CCCCCC

CCCCCC

CCCCCC

GGGGGG

GGGGGG

CCCCCC

GGGGGG

GGGGGG

*To cut the DNA using the same

or different restriction enzymes

that produce 3’ sticky end.

*To add a homopolymer tail to

3’end using terminal transferase

dCTP terminal transferase

*Increasing ligation efficiency

*Decreasing self-reconnection

*2-direction insertion

*no easy way of retrieving the

insert

Generate restriction

site by PCR

123

引物设计时酶切位点的保护碱基表

124

The Taq polymerase has a nontemplate-dependent

terminal transferase activity that adds a single

deoxyadenosine (A) to the 3'-end of the PCR products

125

TOPO cloning is a molecular biology technique in which

DNA fragments amplified by Taq polymerase are cloned into

specific vectors without the requirement for DNA ligases.

Blunt-end ligation

Recombinant

DNA*to cut the DNA using

the same or different

enzymes that produce

blunt ends or sticky

ends that are trimed

to blunt ends by

exonuclease.

*more difficult ligation

*2-direction insertion

*self-reconnection

*no easy way of

retrieving the insert

T4 DNA ligase

vector

Ligation between

sticky and blunt ends

*Introduction of recombinant DNA into

recipient cells (host cells) is called

transformation

*Introduction of foreign DNA changes

(transforms) properties of the organism (host

cells).

Transformation: recombinant

plasmids into E.coli

*Special treatment makes cells competent -

capable of accepting foreign DNA.

*Usually, these treatments make cell membrane

more permeable for a DNA molecule.

0-4℃

Recombinant DNA + 42 ℃

The treatment of host cells:

1. Low-osmosis plus heat shock

E.coli + 0.1M CaCl2

competent (E.coli) cells

E.coli containing recombinant DNA

Low-osmosis makes cell expanding

Heat makes cell more expanding and

membrane more permeable

2. Electroporation makes holes of cell membrane with high voltage

calcium phosphate transfection

DEAE dextran-mediated transfection

Liposome/ Cationic polymer transfection

electroporation

microinjection

Transfection: recombinant DNA into

eukaryotes

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图：倒置荧光显微镜观察pEGFP-

N1在Hela细胞中的表达效果

装置：电穿孔仪

A：普通电转杯B：装有镀金电极的Neon移液器吸头

Neon电转染系统

Microinjection system

DNA microinjection

137

Infection:

recombinant

viral into

eukaryotes

When competent cells are mixed with DNA, some

cells (actually, very few) become transformed:

they acquire recombinant vector DNA.

Pseudo-positive positive

Selection or screening

1. Selection according to the genetic markers

Antibiotic resistance: ampr , tetr , neor

Insertional inactivation

Marker rescue: β-galactosidase

Packaging properties of bacteriophage

140

2. Sequence specificity selction

Restriction map

PCR

Nucleic acid hybridization

DNA sequencing

3. Protein detection

All cells in

the clone are

genetically

identical

In each colony all cells

are descendants of one

transformed cell.

After transformation, cells are plated onto agar medium that

contains selective antibiotic: only transformed cells, that

acquire antibiotic resistance gene on the vector plasmid, will

survive and form colonies. All the untransformed cells will die.

1. Selection according to the genetic markers

antibiotic selection

Plated the cells on the

medium with amp or tet,

only transformed cells can

survive to become clones

Selection according to antibiotic resistance

EcoRI EcoRI

Insertional inactivation

The colonies surviving

only in tet, not in amp

are positive clones

phenotype: AmpsTetr

145

146

Marker rescue: Blue /white selection

148

BamHI

149

IPTG

150

Packaging

properties of

bacteriophage

RE map identification

2. Sequence specificity selction

155

*Incubating and amplifying the bacterial

colonies in proper liquid medium.

*Preparation and purification of plasmid

DNA from the bacteria.

*Cuting the plasmid DNA with the proper

restriction enzymes.

*separating the cut fragments with

electrophoresis.

M：DNA marker

1：purified PCR product of target DNA (2522bp)

2：recombinant plasmid/ Kpn Ⅰ+XhoⅠ(4.8kb+2.5kb）

3：empty plasmid/ Kpn Ⅰ+XhoⅠ （4.8kb）

4： recombinant plasmid /XhoⅠ （7.3kb）

separating the cut fragments with electrophoresis

157

BamHI BamHI

1000bpPst I

200bp 800bp

A BC

Identification of bidirectional

insertion by enzyme cut

BamHI2686bp+1000bp—positive

2686bp—pseudopositive

PstI

BA—200bp + 3486bp

AB—800bp+ 2886bp

4000

3000

2000

1000

200

PCR method

1.Negative control2.Positive control3.Recombinant plasmid4.DNA molecular marker

以提取的重组质粒为模板，利用序列特异性引物扩增插入片段，电泳鉴定正确后，送测序。

Use of hybridization to identify a

clone with a particular DNA

segment. The radioactive DNA

probe hybridizes to

complementary DNA and is

revealed by autoradiography.

Once the labeled colonies have

been identified, the orresponding

colonies on the original agar

plate can be used as a source of

cloned DNA for further study.

In situ hybridization

DNA sequecing

163

3.Protein detection

164

Separation: A specific gene or DNA

fragment and vector DNA that you

selected are separated from their

originals

Cut: Vector DNA and target DNA

are cut at precise locations by proper

restriction enzymes.

Ligation: The target DNA is linked

to the vector DNA covalently by

DNA ligase to form a recombinant

DNA.

Transformation: The recombinant

DNA is introduced into a host cell

that can provide the enzymatic

machinery for DNA replication.

Selection or screening: the host cells

that contain the recombinant DNA

are identified by using some methods

165

Step 6. Gene expression---the production of useful proteins/RNAs

Expression Systems

1. Need a portion of the transcript cDNA or

the gene that encodes the target protein

2. Need a DNA vector (this is usually a

plasmid carrying the cDNA or gene) to

direct transcription

3. Need cellular components that translate,

transcribe, and carry out any post-

translational modifications

Prokaryotic expression system

The bacteria expression system yielded a

high abundance of protein, but the final

protein was improperly folded and thus non-

functional.

Expression vectors of prokaryote should :

1.selection markers ;

2.power promotor;

3. Regulation sequences

4.Polylinker cloning sites

167

Eukaryotic Expression system

Yeast, insect , mammalian cells

Maintains at a high copy number(neo/G418)

High transcription efficiency (hCMV promoter)

High translation efficiency (5'-UTR)

Usually shuttle vectors

168