Download Lecture 6: Gene Expression in Prokaryotes and Eukaryotes

2/21/2011
Lecture 6: Gene Expression in Prokaryotes and Eukaryotes
Suggested readings: Molecular Biotechnology, Chapter 6, 7
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Outline
• Gene expression from strong and regulatable promoters
• Fusion Proteins
• Translation Expression
• Increasing Protein Stability
• DNA Integration into the host chromosome
• Eukaryotic gene expression
• Engineering Mammalian cells for enhanced productivity
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Regulatable Promoters
1. E. coli. lac promoter
• In natural environment, the lac operon allows digestion of lactose to produce glucose (energy source)
• Gene expression is controlled by the concentrations of glucose, lactose and cAMP
• Glucose low  cAMP high; Glucose high  cAMP low
• Gene expression is turned on when glucose is low and lactose is high in the media
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Inducers of the lac promoter
• During recombinant gene expression, lac promoter is induced by synthetic molecule IPTG.
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Regulatable Promoters
2. pL promoter (temperature sensitive)
Turn on protein expression @ 42 C
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Regulatable Promoters
• T7 promoter (pET vectors)
BL21(DE3) cells
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Regulatable Promoters
• Large‐Scale systems (20‐200 L)
– Small culture (1‐5 L), induce protein expression with T shift or adding chemical inducer
– T shift for large system requires time (30‐60 mins) and energy
– Chemical inducer, eg IPTG, can be costly.
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• Regulate gene expression by tryptophan
Achieve foreign protein expression >20% of the cellular protein
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• Expression in other microorganisms
•Different promoters have different efficiency in different organisms.
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Fusion proteins
• Often, small foreign proteins are expressed at very low level due to degradation
• Fuse target protein with a host protein  Improve protein stability
• Fuse target protein with tag  Facilitate protein purification
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Cleavage of Fusion Protein
Precision protease
Intein
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Surface Display
• Facilitate the screening of large cDNA libraries
• M13 phage display system
Target protein is fused to phage protein pIII
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• Bacterial surface display
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Increasing Protein Stability
Stable protein  easier recovery; easier manipulation; larger yield
• Intrinsic protein stability
– Disulfide bonds
– Presence of certain amino acid at the N terminus 
Presence of certain amino acid at the N terminus  protect end
protect end
– Presence of internal PEST sequence (proline, glutamic acid, serine and threonine)  more susceptible to protease degradation
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DNA Integration in the Host Chromosome
• A plasmid imposes a metabolic load on the cell
– High‐copy‐plasmid impose a greater metabolic load than do low‐copy‐plasmid
• Cells lack plasmids grow faster – The number of cell containing plasmid diminishes without selection pressure The number of cell containing plasmid diminishes without selection pressure
(antibiotic or essential metabolite)
– Addition of antibiotic or metabolite can be extremely cost at large‐scale
– Loss of cloned DNA to other microorganism
• DNA integrated into the host chromosome is stable and can be maintained without selection pressure
– Homologous recombination (input DNA share homology with the target region)
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• General principle
Double crossover
Double‐crossover
Single crossover
Single‐crossover
Gene integration is a very rare event ( 1 in a million)  requires selection step to identify the clone containing a transgene
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• Insert gene into a unique predetermined site
1. Integrate marker gene
2. Replace 2
R l
marker gene with target gene
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• Removal of a selectable marker gene
Cre: Type I topoisomerase from P1 bacteriophage that catalyzes site‐specific recombination of DNA between two loxP sites
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Increasing Secretion
• Directing a foreign protein to the periplasm or the growth medium makes its purification easier and less costly, as many fewer proteins are present there than in the cytoplasm
• Secretion of proteins to the periplasm facilitates the disulfide bonds formation
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• Secretion into the periplasm
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Metabolic Load
• Expression of foreign DNA in a host organism changes the metabolism of the organism that may impair normal cellular function
1. Plasmid replication and maintenance requires cellular energy
2. Oxygen limitation in the growth medium
3. Overproduction of foreign protein may deplete the aminoacyl‐tRNA pool
4. The foreign protein may interfere with the function of the host cell
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• Consequence of a metabolic load
1. Decreased cell growth rate
2. Plasmid‐containing cell lose a portion of the plasmid DNA
3. Change in host size and shape
4. Increased translational error (aminoacyl‐tRNA, GTP limiting)
Produce inactive protein
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How to minimize the impact of the metabolic load and optimize the yield of recombinant protein?
1. Use low‐copy‐number rather than high‐copy‐number plasmid
2. Integrate foreign DNA into the chromosomal DNA of the host organism
3. Optimize the codon usage to reflect the codon usage of the host organism
4. Accept a modest level of foreign‐gene expression 24
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Recombinant Protein Production in Eukaryotic Cells
• Posttranslational modification of eukaryotic proteins
– Protein processing: convert inactive form into active form by protease cleavage
Function: 1. Protect host cell from the action of the protein 2. Provide rapid response upon activation
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• Glycosylation: addition of specific sugars to certain amino acids
T: threonine
S: serine
yeast
insect
Mammalian cells
Function: Increase protein stability  protect protein from protease degradation
Target protein to specific location  through interaction with specific receptor
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Eukaryotic Expression Systems
p: t: ESM: orieuk: oriE: Ampr: eukaryotic promoter
termination and polyadenylation
d l d l
signall
euakryotic selection marker
origin of replication in the eukaryotic cells
origin of replication in E. coli
E. coli selectable marker
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• Fungus‐based
– Saccharomyces cerevisiae
– Pichia pastoris Expression System
(methylotropic yeast)
• Baculovirus‐Insect Cell
• Mammalian Cell 28
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Engineering Mammalian Cells for Enhanced Productivity
• Enhance mammalian cell productivity by increasing cell growth, vector stability, gene expression and protein secretion.
Conditions in large‐scale bioreactor can be very stressful to mammalian cells
(d l t d t i t
(depleted nutrients; accumulation of toxic cell waste )
l ti
ft i
ll
t )
1. Inhibit cell death by MDM2
Cultures expressing MDM2 had higher cell densities and delayed cell death compared to nontransfected cells, especially in nutrient‐deprived medium.
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2. Inhibit cell death by reducing toxic byproduct
Oxygen deficient
Oxygen abundant
acidic
Overexpress pyruvate carboxylase in host cells  decrease lactate by 40%
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3. Induce the expression of chaperones
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4. Alter chromatin structure
Addition of an acetyl group to histone can ‘relax’ chromosome condensation, leading to increased transcription of genes in the region.
HAT: histone acetyltransferase
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Discussions
• What are different ways to obtain high levels of expression a foreign protein with rare E. coli codon in E.coli?
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Discussion
• What is the major difference between proteins produced in a yeast, insect, or mammalian cell systems?
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