chapter 8: expression and modification of recombinant proteins i. prokaryotic expression system ii....

Chapter 8 Chapter 8 Expression and Expression and Modification of Recombinant Modification of Recombinant

ProteinsProteins

I Prokaryotic expression system

II Eukaryotic expression system1 Yeast expression2 Insect expression system3 Mammalian expression system

ReferencesReferences

1 Chapter 11 and 13 (Essentials of Molecular Biology)

2 Chapter 10 (An Introduction to Genetic Engineering)

I Prokaryotic I Prokaryotic Expression SystemExpression System

The red boxes represent exons the blue boxes represent the introns and the grey boxes represent the 5rsquo and 3rsquo UTRs

Insert only the ORF into an expression vector

that contains prokaryotic transcriptional and translational regulatory sequences

How do you express this gene in How do you express this gene in bacteriabacteria

Six Step ProcessSix Step Process

① Isolation of gene of interest② Introduction of gene to expression

vector③ Transformation into host cells④ Growth of cells through fermentation⑤ Isolation amp purification of protein⑥ Formulation of protein product

Cloning ProcessCloning Process Gene of interest is cut

out with restriction enzymes (RE)

Host plasmid (circular chromosome) is cut with same REs

Gene is inserted into plasmid and ligated with ligase

New (engineered) plasmid inserted into bacterium (transform)

Fundamentals of Gene Fundamentals of Gene ExpressionExpression

① Prokaryotic and eukaryotic promoters and translation signals are differentthey are not exchangeableYou therefore canrsquot simply put a eukaryotic promoter into bacteria and expect it to function

② Processing also presents a problem for bacterial expression of human mRNAs The eukaryotic ORF must be cloned into the expression vector not the genomic copy

② The genetic code is identical for most bacterial and eukaryotic mRNAsAlthough the code is the same expression levels can be affected by codon frequency which varies between organisms and transcripts

④ Post-translational modifications can be important for protein functionThose modifications might not occur in bacteria The solutionhelliptry expressing in a eukaryotic expression system (viral baculovirus yeast)

Prokaryotic ExpressionProkaryotic ExpressionPros

ConvenientEasy Produce and purify protein using the least expensi

ve and easiest reagents and equipment Best for large scale production of protein

Cons lack many of the immunogenic properties 3D ldquoNativerdquo conformation Lack PTMs (Post translational modifications) need

ed for specific activity

Which VectorWhich VectorPromoters

arabinose systems (pBAD) phage T7 (pET) TrcTac promoters λ PL or PRTags 标签

His6 for metal affinity chromatography (Ni) FLAG epitope tage DYKDDDDK CBP-calmodulin binding peptide (26 residues) E-coilK-coil tags (poly E35 or poly K35) c-myc epitope tag EQKLISEEDL Glutathione-S-transferase (GST) tags Celluluose binding domain (CBD) tags

T7 Promoter

lac Operator

RBS ATG TAG

peptide tag V5 poly-(his)


mutiple cloning site (mcs)

Gene ( with without stop codon )

Fusion ProteinFusion Protein

A Generic Vector

II Eukaryotic Expression II Eukaryotic Expression SystemSystem

1 Yeast expression

2 Insect expression system

3 Mammalian expression system

1 Yeast Expression1 Yeast Expression 1048708 Pros Easier and less expensive than higher eukaryotic cells Rapid growth on inexpensive media Ideal for large-scale production of heterologous protei

ns Often comfortable with genetic manipulation Exhibit near-native conformation PTMs processing Well-defined secretory pathways for extracellular expo

rt of the recombinant gene product Usually safe to use

Cons Lack some PTMs required for specific activities A little more expensive than prokaryote systems Often lack mechanisms for proper folding for some eu

karyote proteins

Yeast Expression Vector

Pichia pastoris

Comments for pPIC9K 9276 nucleotides5acute AOX1 promoter fragment bases 1-9485acute AOX1 primer site bases 855-875a-Factor secretion signal(s) bases 949-1218a-Factor primer site bases 1152-1172Multiple Cloning Site bases 1216-12413acute AOX1 primer site bases 1327-13473acute AOX1 transcription termination (TT)bases 1253-1586HIS4 ORF bases 4514-1980Kanamycin resistance gene bases 5743-49283acute AOX1 fragment bases 6122-6879pBR322 origin bases 7961-7288Ampicillin resistance gene bases 8966-8106

Yeast are single celled eukaryotes Behave like bacteria but have key advan

tages of eukaryotes P pastoris is a methylotrophic甲醇为营养的 yeast that can use methanol甲醇 as its sole carbon source (using alcohol oxidase)

Has a very strong promoter for the alcohol oxidase (AOX) gene (~30 of protein produced when induced)

Cloning in Yeast Cells① Uses a special plasmid that works both in E

coli and Yeast② Once gene of interest is inserted into this pl

asmid it must be linearized (cut open so it isnrsquot circular)

③ Double cross-over recombination event occurs to cause the gene of interest to insert directly into P pastoris chromosome where the old AOX gene used to be

④ Now gene of interest is under control of the powerful AOX promoter

CloningCloning

12

3

4

2 Insect Expression2 Insect ExpressionPros

Produce proteins that has PTMs similar to mammalian systems

Often properly folded and functional Ideal for producing moderate to high levels

of eukaryotic proteins for structure-function assays

Cons Expensive Sometimes proteins are not correctly folded Often not stable

Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)

Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae)

Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell

Baculovirus (AcMNPV) ClBaculovirus (AcMNPV) Cloning Processoning Process

5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA

5rsquo 3rsquoCloned gene

RecombinantAcMNPV DNA

Baculovirus

BacVirus Expression System

pIExtradeInsect Cell Expression Plasmids (Novagen)

Bac-N-BluetradeBaculovirus Expression System (Invitrogen)

3 Mammalian Expression3 Mammalian Expression Pros

Produce protein in the most native and active form Have required PTM machinery to produce active

and useable protein used in mammals Cons Expensive Unstable Low yield and difficulties in purifying recombinant

proteins Limitations on the mechanisms of protein

expression induction Almost always have over expression

HeLa cells in culture

HeLa cells have been cultured continuously for scientific use since they were first taken from the tumor of a woman suffering from cervical cancer in the 1950s They have been utilized for many purposes including the development of a polio vaccine the pursuit of a cure for diseases such as leukemia and cancer and the study of thecellular effects of drugs and radiation

HeLa cells from the Nikon microsc

ope web site

Mitotic HeLa cell stained with anti-Cks1 (red) anti-tubulin (yellow) and DAPI (blue)

JONATHON PINES

REGULATION OF MITOSIS IN

MAMMALIAN CELLS

HeLa Human cells

Expression of (A) -galactosidase and B green fluorescent protein in HeLa cells Cells were transfected in 6-well plates Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection

HeLa cells as you will see them

Expression System Expression System SelectionSelection

Choice depends on size and character of protein

Large proteins (gt100 kD) Choose eukaryote Small proteins (lt30 kD) Choose prokaryote Glycosylation essential Choose baculovirusor杆病毒 mammalian cell culture

High yields low cost Choose E coli Post-translational modifications essential Choo

se yeast baculovirus or other eukaryote

Which VectorWhich Vector Must be compatible with host cell system

(prokaryotic vectors for prokaryotic cells eukaryotic vectors for eukaryotic cells)

Needs a good combination of strong promoters ribosome binding sites termination sequences affinity tag or solubilization sequences multi-enzyme restriction site

Key Parts to a VectorKey Parts to a Vector

Origin of replication (ORI) ndash DNA sequence for DNA polymerase to replicate the plasmid

Selectable marker (Amp or Tet) ndash a gene when expressed on plasmid will allow host cells to survive

Inducible promoter ndash Short DNA sequence which enhances expression of adjacent gene

Multi-cloning site (MCS) ndash Short DNA sequence that contains many restriction enzyme sites

Mammalian ExpressionMammalian Expression Gene initially cloned and plasmid propagated

in bacterial cells Mammalian cells transformed by electroporati

on (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes

Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression amp integration of DHFR and the gene of interest

Methotrexate (MTX) SelecMethotrexate (MTX) Selectiontion

Gene of interest DHFR

Transfectdfhr- cells

Grow inNucleosideFree medium

Culture aColony of cells

Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx



Foreign geneexpressed inhigh level inCHO cells

Mammalian ExpressionSystems

Comments for pcDNAtrade4TO 5078 nucleotidesCMV promoter bases 232-958TATA box bases 804-810Tetracycline operator (2X TetO2) sequences bases 820-859CMV forward priming site bases 769-789Multiple cloning site bases 967-1077BGH reverse priming site bases 1089-1106BGH polyadenylation sequence bases 1095-1319f1 origin bases 1365-1793SV40 promoter and origin bases 1803-2143EM-7 promoter bases 2183-2249Zeocintrade resistance gene bases 2250-2624SV40 early polyadenylation sequence bases 2754-2884pUC origin bases 3267-3937bla promoter bases 4937-5041 (complementary strand)Ampicillin (bla) resistance gene bases 4082-4942 (complementary strand)

Mammalian Cell-line Mammalian Cell-line ExpressionExpression

Sometimes required for difficult-to-express proteins or for ldquocomplete authenticityrdquo (matching glycosylation and sequence)

Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line

Vectors usually use SV-40 virus CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene

SummarySummary1 The mechanism of regulation of gene expres

sion in prokaryotes is greatly different from eukaryotes

2 Regulation of gene expression in prokaryotes is mainly for environmental adaptation

3 Regulation of gene expression in eukaryotes is for cell growth differentiation and development

4 There are four recombinant protein expression systems bacteria (Ecoli) yeast insect and mammalian cells Choose by purpose

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ReferencesReferences

1 Chapter 11 and 13 (Essentials of Molecular Biology)

2 Chapter 10 (An Introduction to Genetic Engineering)



























T7 Promoter

lac Operator

RBS ATG TAG






A Generic Vector


1 Yeast expression







karyote proteins


Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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T7 Promoter

lac Operator

RBS ATG TAG






A Generic Vector


1 Yeast expression







karyote proteins


Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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A Generic Vector


1 Yeast expression







karyote proteins


Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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1 Yeast expression







karyote proteins


Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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karyote proteins


Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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Pichia pastoris










CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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CloningCloning

12

3

4










5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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5rsquo 3rsquo

Transfer vector

Polyhedrin gene

x x

Cloned gene

AcMNPV DNA



Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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Baculovirus












ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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ope web site


JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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JONATHON PINES


MAMMALIAN CELLS

HeLa Human cells

























Grow in005 uM Mtx



Grow in50 uM Mtx

Grow in025 uM Mtx















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Grow in50 uM Mtx

Grow in025 uM Mtx















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chapter 8: expression and modification of recombinant proteins i. prokaryotic expression system ii....

Documents

prokaryotic expression

expression levels

insect expression system

prokaryotic expression

yeast expression pros

yeast slide

generic vector slide

genetic engineering