Transcript Plasmid (E.coli)

Recombinant
DNA
Technology
Dr. Hui LI
Office : S408
Tel: 26538722
[email protected]
Process of cloning
• Isolation of target gene
• Selection and construction of
vectors
• Ligation of target DNA and vector
• Transformation of target gene into
receptor cell
• Screening for recombinant plasmids
• Expressing a cloned gene
Topic 5
Expression of recombinant gene
(Eukaryotes)
Heterologous Protein
Production In Eukaryotic Cells
• Prokaryotic systems are generally
cheaper, but…
• Eukaryotic proteins produced in bacteria
may be
 Unstable or lack biological activity due to lack
of posttranslational modifications or correct
assembly
 Possess unacceptable contaminants after
purification
Posttranslational Modifications
• Correct disulfide bond formation
• Phosphorylation
• Amino acid removal from initial
polypeptide
• O-linked or N-linked glycosylation
 About 30% of eukaryotic proteins are
glycosylated
Generalized Eukaryotic Cloning Vector
• Prokaryotic origin of
replication, selectable
marker
• Eukaryotic origin,
selectable marker
• MCS with eukaryotic
promoter and
transcriptional
terminator/polyadenylati
on signal
Yeast, animal & plant cells as receptor cells
Foreign gene
Promoter
(eukaryotic or virus)
MCS PolyA signal
Terminator
I. Expression in yeasts
1. Expression vectors in yeast
① Cloning and amplification in E.coli(能在E.coli中克隆
和扩增);
Ori
② Selectable marker in E. coli(有大肠杆菌的选择标记);
Ampr、Tetr。
③ Selectable marker in yeasts(有酵母的选择标记);
Leu2+、His+、Ura3+、Trp1+;
④ MCS(克隆位点)。
Dividing Saccharomyces cerevisiae (baker’s yeast) cells
（1） YIp (整合型载体)
由大肠杆菌质粒和酵母的DNA片断（选
择标记）构成。
Ex: PYeleu10:
ColE1(E.coli) Leu 2+ (Yeast)
Yip Vector for
Chromosomal
Insertion
Linear DNA
works Best
• Use LEU2- host strain
• Vector provides
functional gene
• Select on leucine-free
media
• Double recombination
with homologous host
site inserts targeted
portion of recombinant
vector
① Low efficiency of transformation （1-10
transformant/ µgDNA）;
② Impossibility of self-replication in yeast;
载体上只有细菌的复制区，没有
酵母的自主复制区。
③ Integration in the chromosome of yeast
可与受体细胞的染色体DNA同源重组，
随染色体一起复制。
④ Difficult to recover the plasmids from yeast
（2） YRp (复制型载体)
由大肠杆菌质粒和酵母的DNA片断(选择标记
和酵母DNA自主复制顺序ARS）构成。
Plasmid (E.coli) ARS(yeast) Marker(yeast)
ARS
ARS（automously replicating sequence）:
250bp
Conservative
region
ATTTTATATTTA
T
G
T
① High efficiency of transformation （102103 transformant/ µg DNA);
② Easy to extract the plasmids from E.coli
or yeast;
③ “Shuttle vector”（穿梭载体）
既能在大肠杆菌中复制，又能在酵母
细胞中自主复制。
④ not very stable, risk of loss the
foreign gene
（3） YCp (着丝粒质粒)
YRp plasmid + Centromere region of yeast
chromosome (酵母染色体的着丝粒区)
YRp
CEN
① Very stable;
② Exist as single copy (单拷贝存在);
③ Difficult to recover from yeast cell.
（4） YEp(附加体型载体)
Plasmid (E.coli) + 2m plamid + Yeast DNA
Plasmid (E.coli)
2m plasmid Selectable Marker (Yeast)
Ex: pYF92:
pBR322
2m Yeast his 3+
2m plamid: 酿酒酵母的内源质粒，长度是
2m 。含有自主复制起始区ori和STB序列
（使质粒在供体中维持稳定）。
① High efficiency of transformation
（103-105 transformant/ µg DNA);
② High copy number （25-100
copies/cell） 。
③ More stable than YRp
YEp24
2. General process of yeast
transformation and expression
CaCl2、
酶去壁
Leu- 酵母
原生质体 PEG
感受态
提取
大肠杆菌
酵母载体
插入外源基因 转化
Leu营养
转化子克隆生长 缺陷型
培养基筛
选
整合到染色体上，
或独立在酵母细胞内
鉴定克隆
外源基因产物
发酵表达
分离、纯化
Recombinant Proteins
Successfully Produced
in S. cerevisiae
• For a range of
reasons as expressed
previously each of
these represented a
better product than
was obtainable using
a prokaryotic
expression system
Heterologous Protein
Secretion by S. cerevisiae
• Gene must encode leader to pass through
secretory system
 Also aids in correct disulfide bond formation,
proteolytic cleavage of leader, etc. occur
• Over expression of PDI (protein disulfide
isomerase )secretory enzyme also helps
(up to 10X)
Why Other Yeast Species?
• S. cerevisiae sometimes hyperglycosylates
proteins
 Proteins also sometimes retained in
periplasmic space
• S. cerevisiae also produces ethanol at high
cell densities
 Toxic to cells
P. Pastoris (嗜甲烷酵母)
• Highly efficient promoters available
 Tight control (e.g. AOX1 promoter)
 Produce up to 30% of total cell protein by wt.
•
AOX1 (alcohol oxidase for methanol
metabolism) promoter easily turned on by
methanol
• Does not produce ethanol at high cell
density
• Secretes few proteins, simplifying
purification
Baculovirus (杆状病毒)Systems
• Infect invertebrates, including
many insects
• Polyhedron (多角体蛋白) gene
is not essential for life cycle
(protects virus in environment)
• Commonly used with cultured
insect eggs
Baculovirus Vector
• Vector has sequences for expression using polyhedron
gene expression system
• Sequences also present for integration into baculovirus
(AcMNPV) genome via recombination
• Prokaryotic sequences not shown 苜蓿银纹夜蛾细胞核型多角体病毒
（Autographa california multiple
nuclear polyhedrosis virus，
AcMNPV）
Baculovirus Transfer Vector
Done in cell
culture
Screening for
recombinants
tedious by PCR
Examples of Proteins Successfully
Produced by Baculovirus Systems
E. coli Baculovirus Shuttle
Vector - Bacmids
• Shuttle vectors allow ease of transfer between
systems
• Genetic manipulations in one system, expression in
another
Bacmid Construction (2)
• attR and attL are lambda sequences to give high
efficiency and specific transposition
Bacmid Construction (3)
• Bacmid now operates efficiently in both E. coli
and insect cells
Modifying the Insect Cell Host
• Some enzymes simply not present
• Genetic engineering of the host for proper
expression
• Add missing glycosylation enzymes
• Add proteolytic processing enzymes
Mammalian Systems
• Sometimes insect cells simply don’t carry
out proper/necessary glycosylations
• Other processing may also not occur
• Mammalian cell systems are more
expensive by may be required for active
product
Mammalian Expression Vector
• “I” is an intron that enhances expression
• Other signals similar to insect and prokaryotic
vectors
Translation Control Elements
•
•
•
•
•
•
K - Kozak Sequence (equiv. To rbs)
S - for secretion signal peptide
T – tag peptide for purification
P – proteolytic cleavage sequence
SC – stop codon for translation
3’UTR – proper sequences for efficient translation and
mRNA stability (e.g. polyadenylation sequence)
Two Vector Expression System
•Useful for proteins of two different polypeptides
Two Gene Expression Vector
Selectable Markers for Mammalian
Systems
• Most commonly used to select for
transformed cells (killing nonresistant ones)
• Can be used for increasing expression of
heterologous proteins
Selective marker gene systems for mammalian cells
Use of Selectable Markers for
Increasing Heterologous Protein
Production in Mammalian Systems
• Methotrexate (MTX,甲氨蝶呤 ) inhibits
dihydrofolate reductase (DHFR,二氢叶酸还原酶 )
• DHFR- host cell with DHFR gene on cloning
vector (i.e. linked to target gene)
• Gradually increase MTX concentration in culture
• Gene copy number of DHFR and linked target
gene increase to compensate for inhibition of
DHFR (more protein that is less active gives cell
enough metabolic through put to survive)
Thanks, and see you next time!