重组蛋白的表达和修饰
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eukaryote proteins
Yeast Expression Vector
Comments for pPIC9K: 9276 nucleotides 5´ AOX1 promoter fragment: bases 1-948 5´ AOX1 primer site: bases 855-875 a-Factor secretion signal(s): bases 949-1218 a-Factor primer site: bases 1152-1172 Multiple Cloning Site: bases 1216-1241 3´ AOX1 primer site: bases 1327-1347 3´ AOX1 transcription termination (TT): bases 1253-1586 HIS4 ORF: bases 4514-1980 Kanamycin resistance gene: bases 5743-4928 3´ AOX1 fragment: bases 6122-6879 pBR322 origin: bases 7961-7288 Ampicillin resistance gene: bases 8966-8106
* Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)
Cloning in Yeast Cells
① Uses a special plasmid that works both in E.coli and Yeast
* 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
Fusion Protein
Gene ( with / without stop codon )
T7 Promoter
RBS ATG
TAG
lac Operator
peptide tag: V5 / poly-(his) mutiple cloning site (mcs) peptide tag: V5 / poly-(his)
* New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Expression
① Prokaryotic and eukaryotic promoters and translation signals are different...they are not exchangeable
A Generic Vector
II. Eukaryotic Expression System
1. Yeast expression 2. Insect expression system 3. Mammalian expression system
1. Yeast Expression
������ Pros
References
1. Chapter 11 and 13 (Essentials of Molecular Biology)
2. Chapter 10 (An Introduction to Genetic Engineering)
I. Prokaryotic Expression System
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 mRNAs.
Pros
* Convenient/Easy * Produce and purify protein using the least
expensive and easiest reagents and equipment * Best for large scale production of protein
export 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
Chapter 8: Expression and Modification of Recombinant
Proteins
I. Prokaryotic expression system
II. Eukaryotic expression system
1. Yeast expression 2. Insect expression system 3. Mammalian expression system
You therefore can’t simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs.
Promoters * arabinose systems (pBAD), phage T7
(pET), Trc/Tac promoters, λ PL or PR
Tags标签
* His6 for metal affinity chromatography (Ni) * FLAG epitope tage DYKDDDDK * CBP-calmodulin binding peptide (26 residues) * E-coil/K-coil tags (poly E35 or poly K35) * c-myc epitope tag EQKLISEEDL * Glutathione-S-transferase (GST) tags * Celluluose binding domain (CBD) tags
* Easier and less expensive than higher eukaryotic cells * Rapid growth on inexpensive media * Ideal for large-scale production of heterologous
proteins * Often comfortable with genetic manipulation * Exhibit near-native conformation, PTMs, processing * Well-defined secretory pathways for extracellular
* Gene of interest is cut out with restriction enzymes (RE)
* Host plasmid (circular chromosome) is cut with same REs
* Gene is inserted into plasmiห้องสมุดไป่ตู้ and ligated with ligase
Pichia pastoris
* Yeast are single celled eukaryotes
* Behave like bacteria, but have key advantages 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)
Cons
* lack many of the immunogenic properties * 3D “Native” conformation * Lack PTMs (Post translational modifications)
needed for specific activity
Which Vector?
④ Now gene of interest is under control of the powerful AOX promoter
Cloning
1
2
3
4
2. Insect Expression
Pros
* Produce proteins that has PTMs similar to mammalian systems
② Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t 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
vector ③ Transformation into host cells ④ Growth of cells through fermentation ⑤ Isolation & purification of protein ⑥ Formulation of protein product
Cloning Process
How do you express this gene in bacteria?
The red boxes represent exons, the blue boxes represent the introns, and the grey boxes represent the 5’ and 3’ UTRs
Insert only the ORF:
into an expression vector
that contains prokaryotic transcriptional and translational regulatory sequences
Six Step Process
① Isolation of gene of interest ② Introduction of gene to expression
Although 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 function
Those modifications might not occur in bacteria. The solution…try expressing in a eukaryotic expression system (viral, baculovirus, yeast)
Prokaryotic Expression
Yeast Expression Vector
Comments for pPIC9K: 9276 nucleotides 5´ AOX1 promoter fragment: bases 1-948 5´ AOX1 primer site: bases 855-875 a-Factor secretion signal(s): bases 949-1218 a-Factor primer site: bases 1152-1172 Multiple Cloning Site: bases 1216-1241 3´ AOX1 primer site: bases 1327-1347 3´ AOX1 transcription termination (TT): bases 1253-1586 HIS4 ORF: bases 4514-1980 Kanamycin resistance gene: bases 5743-4928 3´ AOX1 fragment: bases 6122-6879 pBR322 origin: bases 7961-7288 Ampicillin resistance gene: bases 8966-8106
* Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)
Cloning in Yeast Cells
① Uses a special plasmid that works both in E.coli and Yeast
* 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
Fusion Protein
Gene ( with / without stop codon )
T7 Promoter
RBS ATG
TAG
lac Operator
peptide tag: V5 / poly-(his) mutiple cloning site (mcs) peptide tag: V5 / poly-(his)
* New (engineered) plasmid inserted into bacterium (transform)
Fundamentals of Gene Expression
① Prokaryotic and eukaryotic promoters and translation signals are different...they are not exchangeable
A Generic Vector
II. Eukaryotic Expression System
1. Yeast expression 2. Insect expression system 3. Mammalian expression system
1. Yeast Expression
������ Pros
References
1. Chapter 11 and 13 (Essentials of Molecular Biology)
2. Chapter 10 (An Introduction to Genetic Engineering)
I. Prokaryotic Expression System
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 mRNAs.
Pros
* Convenient/Easy * Produce and purify protein using the least
expensive and easiest reagents and equipment * Best for large scale production of protein
export 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
Chapter 8: Expression and Modification of Recombinant
Proteins
I. Prokaryotic expression system
II. Eukaryotic expression system
1. Yeast expression 2. Insect expression system 3. Mammalian expression system
You therefore can’t simply put a eukaryotic promoter into bacteria and expect it to function
② Processing also presents a problem for bacterial expression of human mRNAs.
Promoters * arabinose systems (pBAD), phage T7
(pET), Trc/Tac promoters, λ PL or PR
Tags标签
* His6 for metal affinity chromatography (Ni) * FLAG epitope tage DYKDDDDK * CBP-calmodulin binding peptide (26 residues) * E-coil/K-coil tags (poly E35 or poly K35) * c-myc epitope tag EQKLISEEDL * Glutathione-S-transferase (GST) tags * Celluluose binding domain (CBD) tags
* Easier and less expensive than higher eukaryotic cells * Rapid growth on inexpensive media * Ideal for large-scale production of heterologous
proteins * Often comfortable with genetic manipulation * Exhibit near-native conformation, PTMs, processing * Well-defined secretory pathways for extracellular
* Gene of interest is cut out with restriction enzymes (RE)
* Host plasmid (circular chromosome) is cut with same REs
* Gene is inserted into plasmiห้องสมุดไป่ตู้ and ligated with ligase
Pichia pastoris
* Yeast are single celled eukaryotes
* Behave like bacteria, but have key advantages 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)
Cons
* lack many of the immunogenic properties * 3D “Native” conformation * Lack PTMs (Post translational modifications)
needed for specific activity
Which Vector?
④ Now gene of interest is under control of the powerful AOX promoter
Cloning
1
2
3
4
2. Insect Expression
Pros
* Produce proteins that has PTMs similar to mammalian systems
② Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t 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
vector ③ Transformation into host cells ④ Growth of cells through fermentation ⑤ Isolation & purification of protein ⑥ Formulation of protein product
Cloning Process
How do you express this gene in bacteria?
The red boxes represent exons, the blue boxes represent the introns, and the grey boxes represent the 5’ and 3’ UTRs
Insert only the ORF:
into an expression vector
that contains prokaryotic transcriptional and translational regulatory sequences
Six Step Process
① Isolation of gene of interest ② Introduction of gene to expression
Although 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 function
Those modifications might not occur in bacteria. The solution…try expressing in a eukaryotic expression system (viral, baculovirus, yeast)
Prokaryotic Expression