Ch. 7. Recombinant Protein Production in Eukaryotic Cells
I. Yeast Expression Systems II. Insect Cell Expression Systems II. Mammalian Cell Expression Vectors
I. Yeast Expression Systems
II. Insect Cell Expression Systems
II. Mammalian Cell Expression Vectors
Problems With Expression of Eukaryotic Genes In Bacteria
1. Protein instability (proteolysis) 2. Presence of pyrogenic (fever causing) contaminants from bacterial cell membranes in the final product Lipopolysaccharide (endotoxin) in outer membrane of Gram negative bacteria (Ex. E. coli) may contaminate gene product and cause adverse immune response 3. Authentic eukaryotic protein not produced: Bacteria may not perform posttranslational modifications of eukaryotic proteins -Disulfide bond formation for proper protein folding -Proteolytic processing of precursor proteins to produce a functional protein or for secretion from the cell -Reactions that specifically add chemical groups to certain amino acids Acetylation: acetate Acylation: fatty acids Myristylation: addition of 14 carbon chain to N-terminal amino acid Palmitylation: addition of 16 carbon chain to sulfhydryl group of cysteine Carboxylation: -COOH Glycosylation, O- and N-linked carbohydrates Figs. 7.1 & 7.2 Hydroxylation: -OH Phosphorylation: -PO4 Sulfation: -SO4 Functions -Protein structure and stability -Enzyme activity -Anchoring to membranes -Secretion from cell
1. Protein instability (proteolysis)
2. Presence of pyrogenic (fever causing) contaminants from bacterial cell membranes in the final product
Lipopolysaccharide (endotoxin) in outer membrane of Gram negative bacteria (Ex. E. coli) may contaminate gene product and cause adverse immune response
3. Authentic eukaryotic protein not produced:
Bacteria may not perform posttranslational modifications of eukaryotic proteins -Disulfide bond formation for proper protein folding -Proteolytic processing of precursor proteins to produce a functional protein or for secretion from the cell -Reactions that specifically add chemical groups to certain amino acids Acetylation: acetate Acylation: fatty acids Myristylation: addition of 14 carbon chain to N-terminal amino acid Palmitylation: addition of 16 carbon chain to sulfhydryl group of cysteine Carboxylation: -COOH Glycosylation, O- and N-linked carbohydrates Figs. 7.1 & 7.2 Hydroxylation: -OH Phosphorylation: -PO4 Sulfation: -SO4 Functions -Protein structure and stability -Enzyme activity -Anchoring to membranes -Secretion from cell
Bacteria may not perform posttranslational modifications of eukaryotic proteins
-Disulfide bond formation for proper protein folding -Proteolytic processing of precursor proteins to produce a functional protein or for secretion from the cell
-Reactions that specifically add chemical groups to certain amino acids
Acetylation: acetate Acylation: fatty acids Myristylation: addition of 14 carbon chain to N-terminal amino acid Palmitylation: addition of 16 carbon chain to sulfhydryl group of cysteine Carboxylation: -COOH Glycosylation, O- and N-linked carbohydrates Figs. 7.1 & 7.2 Hydroxylation: -OH Phosphorylation: -PO4 Sulfation: -SO4
Acetylation: acetate Acylation: fatty acids
Myristylation: addition of 14 carbon chain to N-terminal amino acid Palmitylation: addition of 16 carbon chain to sulfhydryl group of cysteine
Myristylation: addition of 14 carbon chain to N-terminal amino acid
Palmitylation: addition of 16 carbon chain to sulfhydryl group of cysteine
Carboxylation: -COOH Glycosylation, O- and N-linked carbohydrates Figs. 7.1 & 7.2
Hydroxylation: -OH
Phosphorylation: -PO4
Sulfation: -SO4
Functions
-Protein structure and stability -Enzyme activity -Anchoring to membranes -Secretion from cell
-Protein structure and stability
-Enzyme activity
-Anchoring to membranes
-Secretion from cell
Generalized Eukaryotic Expression Vector
See Fig. 7.3
1. Cloning site with:
-Eukaryotic promoter -Transcription termination sequence -Sequence signaling polyadenylation of mRNA
-Eukaryotic promoter
-Transcription termination sequence
-Sequence signaling polyadenylation of mRNA
2/3. Eukaryotic and prokaryotic selectable markers and ori
Designed to be a shuttle vector for moving genes from cells of one species to cells of another species
Ex. from E. coli to Saccharomyces cerevisiae 1. Use vector to clone cDNA of gene with E. coli host 2. Move recombinant vector to eukaryotic host for expression 3. Host cell expresses the gene and performs posttranslational modifications of gene product
Ex. from E. coli to Saccharomyces cerevisiae
1. Use vector to clone cDNA of gene with E. coli host
2. Move recombinant vector to eukaryotic host for expression
3. Host cell expresses the gene and performs posttranslational modifications of gene product
Yeast Expression Systems
Saccharomyces cerevisae Well known genetics and physiology Generally Recognized As Safe (GRAS) by Food and Drug Administration Used in baking and brewing
Saccharomyces cerevisae
Well known genetics and physiology Generally Recognized As Safe (GRAS) by Food and Drug Administration Used in baking and brewing
Well known genetics and physiology
Generally Recognized As Safe (GRAS) by Food and Drug Administration
Used in baking and brewing
Pichia pastoris AOX1 (alcohol oxidase) allows growth on methanol AOX1 gene has very strong promoter induced by methanol
Pichia pastoris
AOX1 (alcohol oxidase) allows growth on methanol AOX1 gene has very strong promoter induced by methanol
AOX1 (alcohol oxidase) allows growth on methanol
AOX1 gene has very strong promoter induced by methanol
YEps Yeast episomal plasmids High copy number May be lost during large-scale industrial production YIps Yeast integrating vectors Prevents loss of cloned gene(s) Have low gene dosage and low expression levels YACS. Yeast artificial chromosomes Stably maintained as chromosome in host Capable of containing large amounts of cloned DNA Not yet used for expression of commercial proteins
YEps Yeast episomal plasmids
High copy number May be lost during large-scale industrial production
High copy number
May be lost during large-scale industrial production
YIps Yeast integrating vectors
Prevents loss of cloned gene(s) Have low gene dosage and low expression levels
Prevents loss of cloned gene(s)
Have low gene dosage and low expression levels
YACS. Yeast artificial chromosomes
Stably maintained as chromosome in host Capable of containing large amounts of cloned DNA Not yet used for expression of commercial proteins
Stably maintained as chromosome in host
Capable of containing large amounts of cloned DNA
Not yet used for expression of commercial proteins
Saccharomyces cerevisiae Expression System
See Fig. 7.7
1. Yeast origin of replication 2. Eukaryotic selectable marker 3. Yeast promoter and termination-polyadenylation sequence 4. Prokaryotic origin of replication 5. Prokaryotic selectable marker
1. Yeast origin of replication
2. Eukaryotic selectable marker
3. Yeast promoter and termination-polyadenylation sequence
4. Prokaryotic origin of replication
5. Prokaryotic selectable marker
Yeast strain is a LEU- mutant (defective for leucine biosynthesis) --requires leucine in growth medium Vector is selected for (maintained) in mutant cells grown on a medium lacking leucine
Yeast strain is a LEU- mutant (defective for leucine biosynthesis) --requires leucine in growth medium
Vector is selected for (maintained) in mutant cells grown on a medium lacking leucine
Pichia pastoris Integrating Expression System
YIp vector See Fig. 7.8 and 7.9 Cloned DNA is flanked by th 5' and 3' ends of the AOX1 gene on the integrating vector The cloned DNA is integrated into the chromosomal AOX1 gene of the host cell by homologous recombination Eukaryotic selectable marker = HIS4 How would you select for yeast cells containing this vector?
YIp vector
See Fig. 7.8 and 7.9
Cloned DNA is flanked by th 5' and 3' ends of the AOX1 gene on the integrating vector
The cloned DNA is integrated into the chromosomal AOX1 gene of the host cell by homologous recombination
Eukaryotic selectable marker = HIS4
How would you select for yeast cells containing this vector?
Cultured Insect Cell Expression Systems
For expression of eukaryotic gene products that are not correctly postranslationally modified by yeast Vectors derived from baculoviruses which infect insect cells Fig. 7.10 (Latin baculum, rod or stick) Autographa californica (Alfalfa Looper) multiple nuclear polyhedrosis virus (AcMNPV) Infects several insect species and cultured insect cells
For expression of eukaryotic gene products that are not correctly postranslationally modified by yeast
Vectors derived from baculoviruses which infect insect cells Fig. 7.10
(Latin baculum, rod or stick)
Autographa californica (Alfalfa Looper) multiple nuclear polyhedrosis virus (AcMNPV)
Infects several insect species and cultured insect cells
Polyhedrin: a viral protein, gene has a strong promoter Expression is induced during late stages of infection of insect cells
Polyhedrin: a viral protein, gene has a strong promoter
Expression is induced during late stages of infection of insect cells
Polyhedron: composed of polyhedrin with imbedded AcMNPV Released after lysis of insect cell Protects the virus from environmental conditions until eaten by another caterpillar
Polyhedron: composed of polyhedrin with imbedded AcMNPV
Released after lysis of insect cell Protects the virus from environmental conditions until eaten by another caterpillar
Released after lysis of insect cell
Protects the virus from environmental conditions until eaten by another caterpillar
Use of an Insect Cell Expression System
See Fig. 7.11 and 7.12
Two vectors are involved
1. Transfer vector: E. coli plasmid vector for transfer of cloned gene to the baculovirus Contains virus DNA for transfer via recombination
1. Transfer vector: E. coli plasmid vector for transfer of cloned gene to the baculovirus
Contains virus DNA for transfer via recombination
2. Baculovirus expression vector: for expression by cultured insect cells
Both vectors are introduced into cultured insect cells by cotransfection
Homologous recombination between the transfer vector and the baculovirus expression vector transfers the cloned gene to the expression vector Virions form recombinant Baculovirus DNA are isolated form plaques of insect cells grown on plates. Recombinant virons containing the target gene are then used to infect cultured insect cells for expression of the target gene
Homologous recombination between the transfer vector and the baculovirus expression vector transfers the cloned gene to the expression vector
Virions form recombinant Baculovirus DNA are isolated form plaques of insect cells grown on plates.
Recombinant virons containing the target gene are then used to infect cultured insect cells for expression of the target gene
Generalized Mammalian Cell Expression Systems
See Fig. 7.16
1. Eukaryotic ori from animal virus; E.g. SV40 (Simian virus 40) 2. Eukaryotic promoter and transcription termination-polyadenylation sequences (from animal viruses or mammalian genes) 3. Eukaryotic selectable marker (Neomycin resistance, dihydrofolate reductase or glutamine synthetase gene) 4. Multiple cloning site 5. Prokaryotic ori 6. Prokaryotic selectable marker (bla, ampicillin resistance gene) 7. Translation control elements 5' and 3' untranslated regions that increase efficiency of translation and for mRNA stability Kozak sequence for initiation of translation Stop codon
1. Eukaryotic ori from animal virus; E.g. SV40 (Simian virus 40)
2. Eukaryotic promoter and transcription termination-polyadenylation sequences (from animal viruses or mammalian genes)
3. Eukaryotic selectable marker (Neomycin resistance, dihydrofolate reductase or glutamine synthetase gene)
4. Multiple cloning site
5. Prokaryotic ori
6. Prokaryotic selectable marker (bla, ampicillin resistance gene)
7. Translation control elements
5' and 3' untranslated regions that increase efficiency of translation and for mRNA stability Kozak sequence for initiation of translation Stop codon
5' and 3' untranslated regions that increase efficiency of translation and for mRNA stability
Kozak sequence for initiation of translation
Stop codon
E..g. Chinese Hamster ovary cells, baby hamster kidney cells
Expression of Multimeric Proteins in Mammalian Cells
Ex. hemoglobin: a2b2 heterotetramer
1. Two gene (double cassette) expression vector.
Each gene is placed under the control of its on promoter and polyadenylation/transcription termination sequence Two transcripts are produced and translated separately Expression of both genes may not be balanced, ratio of protein chains may not be be correct for formation of functional multemeric protein
Each gene is placed under the control of its on promoter and polyadenylation/transcription termination sequence
Two transcripts are produced and translated separately
Expression of both genes may not be balanced, ratio of protein chains may not be be correct for formation of functional multemeric protein
2. Bicistronic expression vector.
Both genes can be place under the control of the same promoter and polyadenylation/transcription termination sequence The genes are separated by an internal ribosome entry site (IRES) One transcript is produced Translation initiated from the 5'-end of the message produces the first protein chain and translation initiated from the internal IRES produces the second chain IRES Sequence of ~450 nucleotides Allows G-cap independent translation of mRNA Present on some picornaviruses (positive strand RNA viruses) Ex. poliovirus, foot-in-mouth disease virus
Both genes can be place under the control of the same promoter and polyadenylation/transcription termination sequence
The genes are separated by an internal ribosome entry site (IRES)
One transcript is produced
Translation initiated from the 5'-end of the message produces the first protein chain and translation initiated from the internal IRES produces the second chain
IRES
Sequence of ~450 nucleotides
Allows G-cap independent translation of mRNA
Present on some picornaviruses (positive strand RNA viruses)
Ex. poliovirus, foot-in-mouth disease virus
Transformation of yeast cells (uptake of naked DNA)
1. Protoplasts: yeast cell wall removed by digestion with enzymes 2. Chemical competence: treatment of cells with lithium acetate 3. Electroporation
1. Protoplasts: yeast cell wall removed by digestion with enzymes
2. Chemical competence: treatment of cells with lithium acetate
3. Electroporation
Transfection of insect and animal cells
(Term used for animal cells because transformation is used to mean that cell have become cancerous) 1. Coprecipitation of DNA with calcium phosphate, CaHPO4 2. Lipofection DNA is incorporated into lipid vesicles that fuse with cell membrane 3. Microprojectile bombardment (Biolistics) DNA is coated onto small metal particles and fired into the cells 4. Microinjection DNA is manually injected directly into cells --not easy to do
(Term used for animal cells because transformation is used to mean that cell have become cancerous)
1. Coprecipitation of DNA with calcium phosphate, CaHPO4
2. Lipofection
DNA is incorporated into lipid vesicles that fuse with cell membrane
3. Microprojectile bombardment (Biolistics)
DNA is coated onto small metal particles and fired into the cells
4. Microinjection
DNA is manually injected directly into cells --not easy to do
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