Ch 6. Manipulation of Gene Expression in Prokaryotes
Transcription Translation Gene dosage Metabolic load
Transcription
Translation
Gene dosage
Metabolic load
I. Factors Affecting Expression
Commercial production of gene products requires very high expression levels
Expression of genes on cloning vectors in E. coli is often low or nonexistent
Factors that affect gene expression can be manipulated to increase the amount of the gene product
1. Move gene to specialized expression vectors and hosts 2. Location of gene (vector or host's chromosome) 3. Gene dosage 4. Transcription -Promoter and terminator sequences -Regulatory genes and sequences 5. Translation -Ribosome binding site (Shine-Delgarno sequence) -Codon optimization to match host's codon bias 6. Final location of gene product -Cytoplasmic or extracellular (secreted out of cell) 7. Protein stability -Degradation by host proteases -Formation of insoluble aggregates
1. Move gene to specialized expression vectors and hosts
2. Location of gene (vector or host's chromosome)
3. Gene dosage
4. Transcription
-Promoter and terminator sequences -Regulatory genes and sequences
5. Translation
-Ribosome binding site (Shine-Delgarno sequence) -Codon optimization to match host's codon bias
6. Final location of gene product
-Cytoplasmic or extracellular (secreted out of cell)
7. Protein stability
-Degradation by host proteases -Formation of insoluble aggregates
II. Promoters and regulation
Promoters must be present for transcription of a gene by RNA polymerase
RNA polymerase binds to specific nucleotide sequences of promoters
Promoter ~17 nucleotides
Promoter
~17 nucleotides
-------TTGACA----------------TATAAT----------------
-35 ................................. -10 ...........+1 (first nucleotide of transcript)
A gene promoter with the above consensus sequence is strong in E. coli
Strong, regulatable promoters are desirable for controlling expression of cloned genes
Promoters of genes from other organisms may have a different sequence and spacing
The promoter of a gene cloned from an organism may not be recognized by the host's RNA polymerase Transcription of the cloned gene would be low May want to change the promoter to one that is strong in the host you are using
The promoter of a gene cloned from an organism may not be recognized by the host's RNA polymerase
Transcription of the cloned gene would be low
May want to change the promoter to one that is strong in the host you are using
Regulated promoters allow expression of cloned genes to be controlled
Gene product can be produced when conditions are optimal for high-level expression Helps to prevent loss of expression vector from cell during growth High-level expression imposes a metabolic load on the cell Cells without plasmids or that aren't expressing a foreign gene grow faster and take over the culture Some gene products are toxic and kill the cell Grow culture to high cell density first, then induce expression
Gene product can be produced when conditions are optimal for high-level expression
Helps to prevent loss of expression vector from cell during growth
High-level expression imposes a metabolic load on the cell Cells without plasmids or that aren't expressing a foreign gene grow faster and take over the culture
High-level expression imposes a metabolic load on the cell
Cells without plasmids or that aren't expressing a foreign gene grow faster and take over the culture
Some gene products are toxic and kill the cell
Grow culture to high cell density first, then induce expression
Promoters from negatively regulated operons are commonly used to control expression of cloned genes
Expression is induced by IPTG or lactose IPTG is an artificial inducer, used for screeing libraries but it is too expensive for large scale industrial use Lactose is the natural inducer and is much cheaper for production of commercial products
Expression is induced by IPTG or lactose
IPTG is an artificial inducer, used for screeing libraries but it is too expensive for large scale industrial use
Lactose is the natural inducer and is much cheaper for production of commercial products
Expression is repressed by the amino acid tryptophan
Fig. 6.2 Expression is induced by shift to high temperature which inactivates the repressor protein
Fig. 6.2
Expression is induced by shift to high temperature which inactivates the repressor protein
III. Specialized expression vectors
The promoter/operators and regulatory genes of different operons can be combined or they can be placed on different plamids or on the chromosome to engineer useful regulatory systems.
Ex. Expression vector pCP3
See Table 6.1 and Fig. 6.4 Grow host cells at 28OC to high cell density Shift temp. to 42OC to induce expression, also increases plasmid copy number 10 X. However, raising temp. in large industrial fermentors takes time and a lot of energy =$$$$$
See Table 6.1 and Fig. 6.4
Grow host cells at 28OC to high cell density
Shift temp. to 42OC to induce expression, also increases plasmid copy number 10 X.
However, raising temp. in large industrial fermentors takes time and a lot of energy =$$$$$
A dual plasmid system was devised to allow induction of the l PL promoter, without raising the temperature, by addition of tryptone (a cheap source of tryptophan) to the fermentation medium
See Fig. 6.5 Host chromosome contains 1 copy of trpR Plasmid 1 has a low copy number and has cI under control of Ptrp Plasmid 2 has cloned gene under control of PL The genes for the repressor proteins have a low copy number so that too much of the repressor protein isn't produced, but the target gene is on a high copy number plasmid to maxamize expression after induction.
See Fig. 6.5
Host chromosome contains 1 copy of trpR
Plasmid 1 has a low copy number and has cI under control of Ptrp
Plasmid 2 has cloned gene under control of PL
The genes for the repressor proteins have a low copy number so that too much of the repressor protein isn't produced, but the target gene is on a high copy number plasmid to maxamize expression after induction.
IV. Gene dosage effects on expression
Increasing the number of copies of a cloned gene can increase expression
1. Use a high copy number plasmid as an expression vector But this may impose a metabolic load and slow cell growth Cells without vector (and the target gene) will grow faster dominate the culture See Table 6.6 Expression of the gene will be lower than expected 2. Place several copies of target gene on the vector (Tandem gene arrays) See Fig. 6.14 Another approach to increase gene dosage Low copy number vectors can be used, decreasing the metabolic load imposed by high copy number plasmids However: All copies should be unidirectional and in the correct orientation for transcription Too many copies may be unstable, causing rearrangement and deletion of the genes
1. Use a high copy number plasmid as an expression vector
But this may impose a metabolic load and slow cell growth Cells without vector (and the target gene) will grow faster dominate the culture See Table 6.6 Expression of the gene will be lower than expected
But this may impose a metabolic load and slow cell growth
Cells without vector (and the target gene) will grow faster dominate the culture
See Table 6.6
Expression of the gene will be lower than expected
2. Place several copies of target gene on the vector (Tandem gene arrays)
See Fig. 6.14 Another approach to increase gene dosage Low copy number vectors can be used, decreasing the metabolic load imposed by high copy number plasmids However: All copies should be unidirectional and in the correct orientation for transcription Too many copies may be unstable, causing rearrangement and deletion of the genes
See Fig. 6.14
Another approach to increase gene dosage
Low copy number vectors can be used, decreasing the metabolic load imposed by high copy number plasmids
However:
All copies should be unidirectional and in the correct orientation for transcription Too many copies may be unstable, causing rearrangement and deletion of the genes
All copies should be unidirectional and in the correct orientation for transcription
Too many copies may be unstable, causing rearrangement and deletion of the genes
Construction of Tandem Gene Arrays
1. Cut the cloned target gene out of the vector with a restriction enzyme 2. Ligate several copies of the target gene together 3. Insert the tandem gene array into an expression vector and use to transform host cells 4. Screen clones for high expression levels and stability All copies should be unidirectional and in the correct orientation for transcription Too many copies may be unstable, causing rearrangement and deletion of the genes
1. Cut the cloned target gene out of the vector with a restriction enzyme
2. Ligate several copies of the target gene together
3. Insert the tandem gene array into an expression vector and use to transform host cells
4. Screen clones for high expression levels and stability
Factors Affecting Translation of a Cloned Gene
1. Ribosome binding site (RBS) of the transcript -Sequence should be complementary to a sequence present on the 16S rRNA -Located at optimal distance from start codon (5-13 nucleotides) -Formation of intrastrand secondary structure should not hide RBS See Fig. 6.13 -Eukaryotic transcripts do not have a RBS One can be provided (as part of the expression vector) for expression in a bacterial host
1. Ribosome binding site (RBS) of the transcript
-Sequence should be complementary to a sequence present on the 16S rRNA -Located at optimal distance from start codon (5-13 nucleotides) -Formation of intrastrand secondary structure should not hide RBS See Fig. 6.13 -Eukaryotic transcripts do not have a RBS One can be provided (as part of the expression vector) for expression in a bacterial host
-Sequence should be complementary to a sequence present on the 16S rRNA
-Located at optimal distance from start codon (5-13 nucleotides)
-Formation of intrastrand secondary structure should not hide RBS
See Fig. 6.13
-Eukaryotic transcripts do not have a RBS
One can be provided (as part of the expression vector) for expression in a bacterial host
2. Codon bias -Codons present in a cloned gene may be rarely used by the host Host may not produce much of the tRNAs specific for the rare codons 1.) Chemically synthesize the cloned gene using codons preferred by host, or 2.) Provide the host with extra copies of the tRNA genes that have anticodons for the host's rarely used codons Ex. E. coli BL21-Codon Plus (Stratagene) Contains extra copies of E. coli tRNA genes for the rarely used codons Arg codons AGA and AGG are rarest codons in E. coli genome Ile (AUA), Leu (CUA) and Pro (CCC) are also rare and affect amount of protein produced in E. coli hosts tRNA Gene Codon Recognized argU AGA/AGG ileY AUA leuW CUA proL CCC
2. Codon bias
-Codons present in a cloned gene may be rarely used by the host Host may not produce much of the tRNAs specific for the rare codons 1.) Chemically synthesize the cloned gene using codons preferred by host, or 2.) Provide the host with extra copies of the tRNA genes that have anticodons for the host's rarely used codons Ex. E. coli BL21-Codon Plus (Stratagene) Contains extra copies of E. coli tRNA genes for the rarely used codons Arg codons AGA and AGG are rarest codons in E. coli genome Ile (AUA), Leu (CUA) and Pro (CCC) are also rare and affect amount of protein produced in E. coli hosts tRNA Gene Codon Recognized argU AGA/AGG ileY AUA leuW CUA proL CCC
-Codons present in a cloned gene may be rarely used by the host
Host may not produce much of the tRNAs specific for the rare codons
1.) Chemically synthesize the cloned gene using codons preferred by host, or
2.) Provide the host with extra copies of the tRNA genes that have anticodons for the host's rarely used codons
Ex. E. coli BL21-Codon Plus (Stratagene) Contains extra copies of E. coli tRNA genes for the rarely used codons Arg codons AGA and AGG are rarest codons in E. coli genome Ile (AUA), Leu (CUA) and Pro (CCC) are also rare and affect amount of protein produced in E. coli hosts
Ex. E. coli BL21-Codon Plus (Stratagene)
Contains extra copies of E. coli tRNA genes for the rarely used codons
Arg codons AGA and AGG are rarest codons in E. coli genome
Ile (AUA), Leu (CUA) and Pro (CCC) are also rare and affect amount of protein produced in E. coli hosts
3. Stability of the transcript -Most mRNA in prokaryotes is rapidly degraded by ribonucleases (RNases), limiting the amount of translation -Increasing the half-life in the cell would allow the mRNA to be translated several times Half-life = amount of time it takes to make one-half of the transcripts untranslatable -Sequences at the 5' and 3' ends influence susceptibility to degradation by RNases -When more is known, expression vectors may be designed that add stabilizing sequences to mRNA transcripts of cloned genes
3. Stability of the transcript
-Most mRNA in prokaryotes is rapidly degraded by ribonucleases (RNases), limiting the amount of translation -Increasing the half-life in the cell would allow the mRNA to be translated several times Half-life = amount of time it takes to make one-half of the transcripts untranslatable -Sequences at the 5' and 3' ends influence susceptibility to degradation by RNases -When more is known, expression vectors may be designed that add stabilizing sequences to mRNA transcripts of cloned genes
-Most mRNA in prokaryotes is rapidly degraded by ribonucleases (RNases), limiting the amount of translation
-Increasing the half-life in the cell would allow the mRNA to be translated several times
-Sequences at the 5' and 3' ends influence susceptibility to degradation by RNases
-When more is known, expression vectors may be designed that add stabilizing sequences to mRNA transcripts of cloned genes
Expression vector pKK233-2
Designed to provide transcriptional and translational signals to cloned genes for high-level expression in E. coli
Fig. 6.17
Optimizing factors to achieve high levels of transcription and translation may not not result in production of large amounts of the target gene product
Factors Affecting Protein Stability
Following translation, a protein may be degraded by cellular proteases
Ex. Amount of time it takes to decrease b-galactosidase activity by one-half (Table 6.4) N-terminal Met, Ser and Ala half-life > 20 hours N-terminal Arg half-life ~2 min. Although Met is the N-terminal amino acid of (almost) all proteins when they are first synthesized, for various reasons Met may not remain as the N-terminus. Met and additional amino acids may be removed resulting in other amino acids at the N-terminus. Amino acids may be added to the N-terminus.
Ex. Amount of time it takes to decrease b-galactosidase activity by one-half (Table 6.4)
N-terminal Met, Ser and Ala half-life > 20 hours N-terminal Arg half-life ~2 min.
N-terminal Met, Ser and Ala half-life > 20 hours
N-terminal Arg half-life ~2 min.
Although Met is the N-terminal amino acid of (almost) all proteins when they are first synthesized, for various reasons Met may not remain as the N-terminus. Met and additional amino acids may be removed resulting in other amino acids at the N-terminus. Amino acids may be added to the N-terminus.
P proline E glutamic acid S serine T threonine Some solutions: -Alter codons near the 5' end of the gene to encode amino acids that provide greater resistance to proteases -Replace PEST codons with codons for amino acids that are not targets for proteases (if the host is eukaryotic) -Ligate DNA of target gene to gene that encodes a stable host protein, express as a fusion protein See Fig. 6.7 (Use of a fusion protein cloning vector)
P proline E glutamic acid S serine T threonine
Some solutions:
-Alter codons near the 5' end of the gene to encode amino acids that provide greater resistance to proteases
-Replace PEST codons with codons for amino acids that are not targets for proteases (if the host is eukaryotic)
-Ligate DNA of target gene to gene that encodes a stable host protein, express as a fusion protein
See Fig. 6.7 (Use of a fusion protein cloning vector)
Fusion proteins can also be designed to make purification easier.
Ex1. Immunoaffinity chromatography (Fig. 6.8) Ex 2. Fusion to polyhistidine (6x His-tagged protein) can be purified with columns containing chelated Ni++ or other divalent metal ions Following purification, the fusion protein may be cleaved at a specific peptide bond to eliminate the peptide tag from the target gene product Cleavage of peptide bonds may be accomplished with chemical hydrolysis or the use of specific proteases 1. Chemical hydrolysis Acidic pH: -Asp- -Pro- Hydroxylamine: -Asn- -Gly- Cyanogen bromide (CNBr): -Met- -Xaa- 2. Proteolytic enzymes Trypsin: -Arg- -Xaa or -Lys- -Xaa Factor Xa: Ile-Glu-Gly-Arg- -Xaa- Enterokinase: -Asp-Asp-Asp-Asp-Lys- -Gly- Cleavage sites are engineered into fusion protein expression vector at the DNA level Ex. pBAD/His (Invitrogen) contains a multiple cloning site immediately downstream of sequence encoding a cleavage site for enterokinase
Ex1. Immunoaffinity chromatography (Fig. 6.8) Ex 2. Fusion to polyhistidine (6x His-tagged protein) can be purified with columns containing chelated Ni++ or other divalent metal ions
Ex1. Immunoaffinity chromatography (Fig. 6.8)
Ex 2. Fusion to polyhistidine (6x His-tagged protein) can be purified with columns containing chelated Ni++ or other divalent metal ions
Following purification, the fusion protein may be cleaved at a specific peptide bond to eliminate the peptide tag from the target gene product
Cleavage of peptide bonds may be accomplished with chemical hydrolysis or the use of specific proteases
1. Chemical hydrolysis
Acidic pH: -Asp- -Pro-
Hydroxylamine: -Asn- -Gly-
Cyanogen bromide (CNBr): -Met- -Xaa-
2. Proteolytic enzymes
Trypsin: -Arg- -Xaa or -Lys- -Xaa
Factor Xa: Ile-Glu-Gly-Arg- -Xaa-
Enterokinase: -Asp-Asp-Asp-Asp-Lys- -Gly-
Cleavage sites are engineered into fusion protein expression vector at the DNA level
Ex. pBAD/His (Invitrogen) contains a multiple cloning site immediately downstream of sequence encoding a cleavage site for enterokinase
Over expression may cause a protein to aggregate and form inclusion bodies inside the cell
-Insoluble and usually nonfunctional -Often occur when an eukaryotic gene is overexpressed in a prokaryotic host Why? E. coli cytoplasm is different from that of eukaryotic cells pH Ionic strength Oxidation/reduction potential Helper proteins may not be present or in short supply in host Prolyl cis/tans isomerase Disulfide isomerase Chaperones and foldases -The protein may not be correctly folded, causing it to aggregate with itself -Inclusion bodies may also form if expression is too rapid for proper folding May need to slow rate of protein synthesis by reducing the temperature during expression
-Insoluble and usually nonfunctional
-Often occur when an eukaryotic gene is overexpressed in a prokaryotic host
Why?
E. coli cytoplasm is different from that of eukaryotic cells pH Ionic strength Oxidation/reduction potential Helper proteins may not be present or in short supply in host Prolyl cis/tans isomerase Disulfide isomerase Chaperones and foldases
E. coli cytoplasm is different from that of eukaryotic cells
pH Ionic strength Oxidation/reduction potential Helper proteins may not be present or in short supply in host Prolyl cis/tans isomerase Disulfide isomerase Chaperones and foldases
pH
Ionic strength
Oxidation/reduction potential
Helper proteins may not be present or in short supply in host
Prolyl cis/tans isomerase Disulfide isomerase Chaperones and foldases
Prolyl cis/tans isomerase
Disulfide isomerase
Chaperones and foldases
-The protein may not be correctly folded, causing it to aggregate with itself
-Inclusion bodies may also form if expression is too rapid for proper folding
May need to slow rate of protein synthesis by reducing the temperature during expression
Secretion of protein into medium makes product recovery (downstream processing) simplier Gram negative bacteria usually do not secrete proteins Outer membrane acts as a barrier to secretion L-form strains that lack a cell wall (and outer membrane) can secrete proteins Gram positive bacteria (Ex. Corynebacterium, Bacillus) lack an outer membrane and often secrete proteins into the medium Most secreted proteins need an N-terminal leader peptide to pass through the cell membrane Secretion of recombinant proteins can be genetically engineered by addition of DNA sequence encoding a leader peptide to the target gene Cloned gene is place dowmstream of the coding sequence of a leader peptide Expression produces a fusion protein containing a leader peptide During translation, the protein is directed through the cell membrane to the exterior of the cell and the leader peptide is removed However, over expression of foreign proteins can jam the secretory pathway
Secretion of protein into medium makes product recovery (downstream processing) simplier
Gram negative bacteria usually do not secrete proteins
Outer membrane acts as a barrier to secretion L-form strains that lack a cell wall (and outer membrane) can secrete proteins
Outer membrane acts as a barrier to secretion
L-form strains that lack a cell wall (and outer membrane) can secrete proteins
Gram positive bacteria (Ex. Corynebacterium, Bacillus) lack an outer membrane and often secrete proteins into the medium
Most secreted proteins need an N-terminal leader peptide to pass through the cell membrane
Secretion of recombinant proteins can be genetically engineered by addition of DNA sequence encoding a leader peptide to the target gene
Cloned gene is place dowmstream of the coding sequence of a leader peptide
Expression produces a fusion protein containing a leader peptide During translation, the protein is directed through the cell membrane to the exterior of the cell and the leader peptide is removed
Expression produces a fusion protein containing a leader peptide
During translation, the protein is directed through the cell membrane to the exterior of the cell and the leader peptide is removed
However, over expression of foreign proteins can jam the secretory pathway
Advantages
1.) Cheaper: maintains gene in the host without selection Once integrated, the cloned gene isn't easily lost due to metabolic load because the host must maintain its chromosome Plasmid vectors containing the cloned gene are maintained in host by selective pressure Ex. Addition of expensive antibiotics to the growth medium
1.) Cheaper: maintains gene in the host without selection
Once integrated, the cloned gene isn't easily lost due to metabolic load because the host must maintain its chromosome Plasmid vectors containing the cloned gene are maintained in host by selective pressure Ex. Addition of expensive antibiotics to the growth medium
Once integrated, the cloned gene isn't easily lost due to metabolic load because the host must maintain its chromosome
Plasmid vectors containing the cloned gene are maintained in host by selective pressure
Ex. Addition of expensive antibiotics to the growth medium
2.) Lessens chance of horizontal transfer to other organisms Do not want genetically engineered organisms released into the environment to spread foreign genes to the natural microbial populations by transfer of recombinant plasmids
2.) Lessens chance of horizontal transfer to other organisms
Do not want genetically engineered organisms released into the environment to spread foreign genes to the natural microbial populations by transfer of recombinant plasmids
-Clone the nonessential host gene or part of it and place it on a plasmid vector -Insert the gene to be integrated into the nonessential gene on the vector -Introduce the vector into the host cell -The gene of interest will be incorporated into the host's chromosome through homologous recombination. and the plasmid will be lost in the absence of selection.
-Clone the nonessential host gene or part of it and place it on a plasmid vector
-Insert the gene to be integrated into the nonessential gene on the vector
-Introduce the vector into the host cell
-The gene of interest will be incorporated into the host's chromosome through homologous recombination. and the plasmid will be lost in the absence of selection.
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