Recombinant DNA

Technology

Dr. Hui LIOffice : S408Tel: 26538722lihui80@szu.edu.cn

• 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 　

Process of cloning

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/polyadenylation signal

Yeast, animal & plant cells as receptor cells

Promoter(eukaryotic or virus) MCS PolyA signal Terminator

Foreign gene

① Cloning and amplification in E.coli( 能在 E.coli 中克隆和扩增 );

1. Expression vectors in yeast

I. Expression in yeasts

Leu2+ 、 His+ 、 Ura3+ 、 Trp1+

; ④ MCS( 克隆位点 ) 。

③ Selectable marker in yeasts( 有酵母的选择标记 );

Ori

② Selectable marker in E. coli( 有大肠杆菌的选择标记 );

Ampr 、 Tetr 。

Dividing Saccharomyces cerevisiae (baker’s yeast) cells

由大肠杆菌质粒和酵母的 DNA 片断（选择标记）构成。

ColE1(E.coli) Leu 2+ (Yeast)

Ex: PYeleu10:

（ 1 ） YIp ( 整合型载体 )

Yip Vector for Chromosomal

Insertion• 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

Linear DNA works Best

① Low efficiency of transformation （ 1-10 transformant/ µgDNA ） ;

载体上只有细菌的复制区，没有酵母的自主复制区。

可与受体细胞的染色体 DNA 同源重组，随染色体一起复制。

② Impossibility of self-replication in yeast;

③ Integration in the chromosome of yeast

④ Difficult to recover the plasmids from yeast

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)

（ 2 ） YRp ( 复制型载体 )

Marker(yeast)

ARS

ARS （ automously replicating sequence ） :

ATTTTATATTTAT G T

250bp

Conservative region

① High efficiency of transformation （ 102-103 transformant/ µg DNA);

④ not very stable, risk of loss the foreign gene

② Easy to extract the plasmids from E.coli or yeast;

既能在大肠杆菌中复制，又能在酵母细胞中自主复制。

③ “Shuttle vector” （穿梭载体）

YRp plasmid + Centromere region of yeast chromosome ( 酵母染色体的着丝粒区 )

YRp CEN

（ 3 ） YCp ( 着丝粒质粒 )

③ Difficult to recover from yeast cell.

① Very stable;② Exist as single copy ( 单拷贝存在 );

Plasmid (E.coli) + 2m plamid + Yeast DNA

Plasmid (E.coli) Selectable Marker (Yeast) 2m plasmid

Ex: pYF92:

pBR322 2m Yeast his 3+

（ 4 ） YEp( 附加体型载体 )

2m plamid: 酿酒酵母的内源质粒，长度是2m 。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

Leu- 酵母 原生质体 感受态酶去壁 CaCl2 、

PEG

整合到染色体上，或独立在酵母细胞内

转化

Leu 营养缺陷型培养基筛选

转化子克隆生长

酵母载体大肠杆菌提取

插入外源基因

鉴定克隆 发酵表达 外源基因产物分离、纯化

2. General process of yeast transformation and expression

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 cellculture

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!