Chapter 8. Directed Mutagenesis and Protein Engineering.
I. Oligonucleotide Directed Mutagenesis II. Random Mutagenesis III. Error-prone PCR IV. DNA shuffling V. Protein Engineering
I. Oligonucleotide Directed Mutagenesis
II. Random Mutagenesis
III. Error-prone PCR
IV. DNA shuffling
V. Protein Engineering
Effects of Mutations* on Protein Properties
*A change in the nucleotide sequence of a gene.
Ex. 5'-----TTT----- encodes phenylalanine, a hydrophobic amino acid 5'-----TAT----- a point mutation that changes the codon to one that encodes tyrosine, a polar amino acid The single amino acid change may alter the protein's structure or activity (or may have no effect) Millions of years of evolution have selected for the wild type amino acid sequence of a protein Most mutations have a negative effect, rarely are beneficial to host (or biotechnologist) Difficult to predict the effects of most mutations Exceptions: (these aren't the ones you want to make usually) Silent mutations -no effect Nonsense mutations -create a stop codon within the coding region, causes expression of a truncated protein, not functional Insertions/deletions -altered reading frame, produces wrong amino acid sequence So, making improvements or designing proteins for biotechnological applications is not easy
Ex.
5'-----TTT----- encodes phenylalanine, a hydrophobic amino acid
5'-----TAT----- a point mutation that changes the codon to one that encodes tyrosine, a polar amino acid
The single amino acid change may alter the protein's structure or activity (or may have no effect)
Millions of years of evolution have selected for the wild type amino acid sequence of a protein
Most mutations have a negative effect, rarely are beneficial to host (or biotechnologist)
Difficult to predict the effects of most mutations
Exceptions: (these aren't the ones you want to make usually) Silent mutations -no effect Nonsense mutations -create a stop codon within the coding region, causes expression of a truncated protein, not functional Insertions/deletions -altered reading frame, produces wrong amino acid sequence
Exceptions: (these aren't the ones you want to make usually)
Silent mutations -no effect
Nonsense mutations -create a stop codon within the coding region, causes expression of a truncated protein, not functional
Insertions/deletions -altered reading frame, produces wrong amino acid sequence
So, making improvements or designing proteins for biotechnological applications is not easy
Uses for Directed Mutagenesis
I. Increase expression levels
1. Alter sequence of promoters, operators, terminators, ribosome-binding site 2. Change codons of gene to those preferred by host
1. Alter sequence of promoters, operators, terminators, ribosome-binding site
2. Change codons of gene to those preferred by host
II. Engineering proteins with desired properties
Enzyme 1. Enzyme activity: Substrate -----------> Product Increase affinity for substrate (lower Km) Increase maximum rate of the reaction (increase Vmax ) Prevent product and other compounds from inhibiting activity Alter substrate specificity Broaden: To include other substrates or Narrow : Eliminate production of unwanted products
Enzyme
1. Enzyme activity: Substrate -----------> Product
Increase affinity for substrate (lower Km) Increase maximum rate of the reaction (increase Vmax ) Prevent product and other compounds from inhibiting activity Alter substrate specificity Broaden: To include other substrates or Narrow : Eliminate production of unwanted products
Increase affinity for substrate (lower Km)
Increase maximum rate of the reaction (increase Vmax )
Prevent product and other compounds from inhibiting activity
Alter substrate specificity
Broaden: To include other substrates or Narrow : Eliminate production of unwanted products
Broaden: To include other substrates or
Narrow : Eliminate production of unwanted products
2. Alter biological activity. Ex. Hormones, immune system effectors, biological pesticides
2. Alter biological activity.
Ex. Hormones, immune system effectors, biological pesticides
3. Protein stability. Increase resistance to: Degradation by proteases Denaturation by organic solvents or extreme temperature and pH
3. Protein stability. Increase resistance to:
Degradation by proteases Denaturation by organic solvents or extreme temperature and pH
Degradation by proteases
Denaturation by organic solvents or extreme temperature and pH
4. Regulatory proteins -- to manipulate expression Ex.. Alter effector specificity
4. Regulatory proteins -- to manipulate expression
Ex.. Alter effector specificity
Oligonucleotide-Directed Mutagenesis
Should know nucleotide sequence of DNA that you want to change Chemically synthesize an oligonucleotide with the desired change Use as a primer for synthesis of the gene using a DNA polymerase (Klenow fragment)
Should know nucleotide sequence of DNA that you want to change
Chemically synthesize an oligonucleotide with the desired change
Use as a primer for synthesis of the gene using a DNA polymerase (Klenow fragment)
Oligonucleotide-directed mutagenesis (site-specific mutagenesis) with M13 bacteriophage
M13 is a bacteriophage that infects E. coli
-One of smallest phages -Genome is circular, 6407 nucleotides of single-stranded DNA, encodes only 10 genes, Replication cycle Stage 1. Infection of an E. coli cell occurs when the + (plus) form of single stranded (ss) M13 DNA is injected into the cytoplasm A complementary DNA strand is then synthesized producing a double-stranded (ds) circular molecule (replicative form, RF) Stage 2. RF DNA is replicated to produce 100 to 200 daughter ds RF molecules Stage 3. ds RF produce ss + strand molecules which are coated with a capsid as they exit the cell without causing lysis
-One of smallest phages -Genome is circular, 6407 nucleotides of single-stranded DNA, encodes only 10 genes,
Replication cycle
Stage 1. Infection of an E. coli cell occurs when the + (plus) form of single stranded (ss) M13 DNA is injected into the cytoplasm
A complementary DNA strand is then synthesized producing a double-stranded (ds) circular molecule (replicative form, RF)
Stage 2. RF DNA is replicated to produce 100 to 200 daughter ds RF molecules
Stage 3. ds RF produce ss + strand molecules which are coated with a capsid as they exit the cell without causing lysis
Used as a vector for mutagenesis because it produces single stranded DNA needed for hybridization to mutagenic oligonucleotide primers
See Fig. 8.1 1. Synthesize an oligonucleotide containing the changed sequence Ex. ---ATT--- Wild type sequence (a codon for Ile) ---CTT--- Desired change (Leu) ---GAA--- Mutagenic oligonucleotide 2. Hybridize the mutagenic oligonucleotideto single stranded form of gene cloned into M13 Use low stringency hybridization conditions (low temperature + high ionic strength --see below) 3. Synthesize second strand of DNA with DNA polymerase (Klenow fragment) and dNTPs 4. Seal nick in new strand with T4 DNA ligase 5. Introduce into E. coli where Stage 2 replication produces more ds molecules Semiconservative replication results in 1/2 of ds DNA molecules with the mutation and the other 1/2 with the wild-type sequence 6. After Stage 3 of phage replication, ss + phage are isolated from plaques and screened by hybridization to the original mutagenic oligonucleotid (appropriately labeled for detection) Hybridization is performed under high stringency conditions (high temperature, lower salt) to prevent hybridization to the wild-type sequence
See Fig. 8.1
1. Synthesize an oligonucleotide containing the changed sequence
Ex. ---ATT--- Wild type sequence (a codon for Ile) ---CTT--- Desired change (Leu) ---GAA--- Mutagenic oligonucleotide
---ATT--- Wild type sequence (a codon for Ile)
---CTT--- Desired change (Leu)
---GAA--- Mutagenic oligonucleotide
2. Hybridize the mutagenic oligonucleotideto single stranded form of gene cloned into M13
Use low stringency hybridization conditions (low temperature + high ionic strength --see below)
3. Synthesize second strand of DNA with DNA polymerase (Klenow fragment) and dNTPs
4. Seal nick in new strand with T4 DNA ligase
5. Introduce into E. coli where Stage 2 replication produces more ds molecules
Semiconservative replication results in 1/2 of ds DNA molecules with the mutation and the other 1/2 with the wild-type sequence
6. After Stage 3 of phage replication, ss + phage are isolated from plaques and screened by hybridization to the original mutagenic oligonucleotid (appropriately labeled for detection)
Hybridization is performed under high stringency conditions (high temperature, lower salt) to prevent hybridization to the wild-type sequence
Stringency of DNA Hybridization Conditions
The stringency of the hybridization conditions affects the stregnth of hydrogen bonding between complementary base pairs of two DNA strands. 1. Conditions that do not disrupt hydrogen bonds have low stringency Low hybridization temperature, ~40OC, or high salt concentration Some mismatched basepairs can be present AAGCAAGCATGC TGCGTCCGTGCG
The stringency of the hybridization conditions affects the stregnth of hydrogen bonding between complementary base pairs of two DNA strands.
1. Conditions that do not disrupt hydrogen bonds have low stringency
Low hybridization temperature, ~40OC, or high salt concentration Some mismatched basepairs can be present AAGCAAGCATGC TGCGTCCGTGCG
Low hybridization temperature, ~40OC, or high salt concentration
Some mismatched basepairs can be present
AAGCAAGCATGC TGCGTCCGTGCG
2. Conditions that weaken hydrogen bonds have high stringency High temperature ~65OC, or low salt concentration, or organic solvents (e.g. dimethylformamide) The nucleotide sequences of the two strands must be highly complimentary for hybridization to occur ATGCATGCATGC TACGTACGTACG
2. Conditions that weaken hydrogen bonds have high stringency
High temperature ~65OC, or low salt concentration, or organic solvents (e.g. dimethylformamide) The nucleotide sequences of the two strands must be highly complimentary for hybridization to occur ATGCATGCATGC TACGTACGTACG
High temperature ~65OC, or low salt concentration, or organic solvents (e.g. dimethylformamide)
The nucleotide sequences of the two strands must be highly complimentary for hybridization to occur
ATGCATGCATGC TACGTACGTACG
Oligonucleotide-Directed Mutagenesis Using a Plasmid Vector
This method increases the chances of obtaining the desired mutation in the target
See Fig. 8.3 Introduce mutations into target gene and 2 antibiotic resistance genes (ampicillin and tetracycline) The tetracycline resistance gene protects cells that contain the vector from exposure to tetracycline The ampicillin resistance gene contains a mutation that encodes an inactive form of beta-lactamase Cells containing the vector are sensitive to and killed by ampicillin One oligonucleotide corrects the mutation in the ampicillin resistance gene, and cells that contain the vector become resistant to ampicillin A second oligonucleotide introduces a mutation into the tetracycline resistance gene, making cells that contain it sensitive to tetracycline A third mutagenic oligonucleotide introduces the desired mutation into the target gene After introducing the mutagenized vector into the host, cells that are resistant to ampicillin and sensitive to tetracycline are likely to contain a mutated target gene Vectors with mutations in both antibiotic resistance genes have higher chance of also carrying a mutated target gene
See Fig. 8.3
Introduce mutations into target gene and 2 antibiotic resistance genes (ampicillin and tetracycline)
The tetracycline resistance gene protects cells that contain the vector from exposure to tetracycline The ampicillin resistance gene contains a mutation that encodes an inactive form of beta-lactamase Cells containing the vector are sensitive to and killed by ampicillin One oligonucleotide corrects the mutation in the ampicillin resistance gene, and cells that contain the vector become resistant to ampicillin A second oligonucleotide introduces a mutation into the tetracycline resistance gene, making cells that contain it sensitive to tetracycline A third mutagenic oligonucleotide introduces the desired mutation into the target gene After introducing the mutagenized vector into the host, cells that are resistant to ampicillin and sensitive to tetracycline are likely to contain a mutated target gene Vectors with mutations in both antibiotic resistance genes have higher chance of also carrying a mutated target gene
The tetracycline resistance gene protects cells that contain the vector from exposure to tetracycline
The ampicillin resistance gene contains a mutation that encodes an inactive form of beta-lactamase
Cells containing the vector are sensitive to and killed by ampicillin
One oligonucleotide corrects the mutation in the ampicillin resistance gene, and cells that contain the vector become resistant to ampicillin
A second oligonucleotide introduces a mutation into the tetracycline resistance gene, making cells that contain it sensitive to tetracycline
A third mutagenic oligonucleotide introduces the desired mutation into the target gene
After introducing the mutagenized vector into the host, cells that are resistant to ampicillin and sensitive to tetracycline are likely to contain a mutated target gene
Vectors with mutations in both antibiotic resistance genes have higher chance of also carrying a mutated target gene
PCR-Amplified Oligonucleotide-Directed Mutagenesis
See Fig. 8.4
Perform two separate PCR reactions with two sets of primers
Each primer set introduces the desired nucleotide change but produces linear PCR products with different ends Denaturation, mixing aand ligation of the PCR products produces mutated circular DNA that can be introduced into E. coli by transformation
Each primer set introduces the desired nucleotide change but produces linear PCR products with different ends
Denaturation, mixing aand ligation of the PCR products produces mutated circular DNA that can be introduced into E. coli by transformation
Random Mutagenesis With Degenerate Oligonucleotide Primers
See Fig. 8.5 Mutations are random and not targeted to one specific nucleotide Use if you don't know what amino acid needs to be changed to alter your protein's properties Random changes in the nucleotide sequence of the primer are introduced during chemical synthesis of a pool of degenerate oligonucleotides Must express mutant genes and screen gene products to find the improved version of the protein
See Fig. 8.5
Mutations are random and not targeted to one specific nucleotide
Use if you don't know what amino acid needs to be changed to alter your protein's properties
Random changes in the nucleotide sequence of the primer are introduced during chemical synthesis of a pool of degenerate oligonucleotides
Must express mutant genes and screen gene products to find the improved version of the protein
Random Mutagenesis With Base Analogs
See Fig. 8.7 Two restriction enzymes, ExonucleaseIII and DNA polymerase are used to produce DNA with a nucleotide containing a base analog randomly incorporated into the target DNA Ex. N4-hydroxy cytosine is an analog of cytosine Presence in DNA causes an A-T basepair to G-C basepair transition mutation when the DNA is replicated by the host Ex. ---AAA--- (Lys) to ---GAA--- (Glu) ---TTT--- ...............---CTT---
See Fig. 8.7
Two restriction enzymes, ExonucleaseIII and DNA polymerase are used to produce DNA with a nucleotide containing a base analog randomly incorporated into the target DNA
Ex. N4-hydroxy cytosine is an analog of cytosine
Presence in DNA causes an A-T basepair to G-C basepair transition mutation when the DNA is replicated by the host
---AAA--- (Lys) to ---GAA--- (Glu)
---TTT--- ...............---CTT---
Random Mutagenesis via Error-Prone PCR
Fig. 8.8 DNA polymerase form Thermus aquaticus sometimes incorporates the wrong nucleotide during synthesis of a strand of DNA The enzyme cannot correct mistakes which then become mutations Error rate ~1/10,000 base pairs Error rate can be increased by altering PCR reaction conditions Ex. Addition of Mn2+ and unequal conc. of dNTPs DNA from amplified genes is cloned to create a library of mutant genes Gene products from expression of mutant genes are screened for improved properties
Fig. 8.8
DNA polymerase form Thermus aquaticus sometimes incorporates the wrong nucleotide during synthesis of a strand of DNA
The enzyme cannot correct mistakes which then become mutations
Error rate ~1/10,000 base pairs
Error rate can be increased by altering PCR reaction conditions
Ex. Addition of Mn2+ and unequal conc. of dNTPs
DNA from amplified genes is cloned to create a library of mutant genes
Gene products from expression of mutant genes are screened for improved properties
DNA Shuffling
Fig. 8.10 The sequences of families of related genes are shuffled to create hybrid genes which may encode gene products with improved properties Restriction enzyme sites common to all of the wild type genes are cut to produce fragments that are combined and ligated to produce hydrid genes Ex. a-interferon genes (We will cover this example in Ch. 10)
Fig. 8.10
The sequences of families of related genes are shuffled to create hybrid genes which may encode gene products with improved properties
Restriction enzyme sites common to all of the wild type genes are cut to produce fragments that are combined and ligated to produce hydrid genes
Ex. a-interferon genes (We will cover this example in Ch. 10)
Industrial Enzymes
See Table 8.1 Most enzymes are unsuitable for industrial use because of harsh conditions that denature most proteins High temperature High or low pH Presence of organic solvents or other chemicals About 20 enzymes are used in 90% of all industrial applications
See Table 8.1
Most enzymes are unsuitable for industrial use because of harsh conditions that denature most proteins
High temperature High or low pH Presence of organic solvents or other chemicals
About 20 enzymes are used in 90% of all industrial applications
Engineering Proteins for Industrial Applications
1. Increased protein stability Thermal stability -Add intramolecular covalent (disulfide) bonds by introducing Cys residues Prevents protein unfolding at elevated temperature
1. Increased protein stability
Thermal stability -Add intramolecular covalent (disulfide) bonds by introducing Cys residues Prevents protein unfolding at elevated temperature
Thermal stability
-Add intramolecular covalent (disulfide) bonds by introducing Cys residues Prevents protein unfolding at elevated temperature
-Add intramolecular covalent (disulfide) bonds by introducing Cys residues
Prevents protein unfolding at elevated temperature
-Eliminate Asn and Gln which lose their amino group (deamidate) at high temp.
Resistance to proteolysis -Change N-terminal and PEST amino acids that are targets for proteases (Increasing temperature stability may also increases resistance to proteases)
Resistance to proteolysis
-Change N-terminal and PEST amino acids that are targets for proteases (Increasing temperature stability may also increases resistance to proteases)
2. Preventing formation of insolube aggregates (inclusion bodies) Change Cys involved in intermolecular disulfide bonds to a different amino acid
2. Preventing formation of insolube aggregates (inclusion bodies)
Change Cys involved in intermolecular disulfide bonds to a different amino acid
3. Altering enzyme activity Increase the rate of the reaction Increase affinity for substrate Change specificity for substrate
3. Altering enzyme activity
Increase the rate of the reaction Increase affinity for substrate Change specificity for substrate
Increase the rate of the reaction
Increase affinity for substrate
Change specificity for substrate
Difficult, usually requires information that isn't readily available i. Detailed knowledge of the 3-D structure (solved by X-ray crystallography or nuclear magnetic resonance spectrometry) ii. Knowledge of the reaction mechanism and amino acids involved in catalyzing the reaction
Difficult, usually requires information that isn't readily available
i. Detailed knowledge of the 3-D structure (solved by X-ray crystallography or nuclear magnetic resonance spectrometry) ii. Knowledge of the reaction mechanism and amino acids involved in catalyzing the reaction
Intramolecular Disulfide Bonds: Effect on Thermostability of Lysozyme
See Table 8.2 Cys introduced by oligonucleotide directed mutagenesis Amino acids targeted for change: Close to each other in folded protein Not in or near active site
See Table 8.2
Cys introduced by oligonucleotide directed mutagenesis
Amino acids targeted for change:
Close to each other in folded protein Not in or near active site
Close to each other in folded protein
Not in or near active site
High Temperature Deamidation
Deamidation. At high temps. Asn and Gln in proteins lose their amino groups, producing Asp and Glu, respectively
This change is from a polar uncharged R group to a negatively charged R group which may disrupt: 3-Dimensional conformation Enzyme active site geometry or binding to cofactors Protein-protein interaction
This change is from a polar uncharged R group to a negatively charged R group which may disrupt:
3-Dimensional conformation Enzyme active site geometry or binding to cofactors Protein-protein interaction
3-Dimensional conformation
Enzyme active site geometry or binding to cofactors
Protein-protein interaction
The thermal stability of triose phosphate isomerase was increased using directed mutagenesis to change two Asn residues to Thr and Ile which are not susceptable to deamidation.
See Table 8.3
Eliminating the Requirement for a Metal Cofactor
See Fig. 8.17 and Table 8.5
Subtilisin
Protease added to laundry detergent Requires Ca++ for stability, but detergents have chelators that bind metals
Protease added to laundry detergent
Requires Ca++ for stability, but detergents have chelators that bind metals
To increase stability in your washing machine:
1. Ca++binding domain was deleted 2. Random mutagenesis was used to make changes in 4 regions of the protein
1. Ca++binding domain was deleted
2. Random mutagenesis was used to make changes in 4 regions of the protein
Clones were screened for expression of a thermostable enzyme Seven separate mutations increased stability All 7 were combined in a single gene Enzyme was 10 x more stable than native (wild type) enzyme in the absence of Ca++ and 1.5 x as stable in its presence
Clones were screened for expression of a thermostable enzyme
Seven separate mutations increased stability
All 7 were combined in a single gene
Enzyme was 10 x more stable than native (wild type) enzyme in the absence of Ca++ and 1.5 x as stable in its presence
Lowering Degradation by Proteases
Streptokinase
Produced by Streptococcus Protease that dissolves blood clots, used to treat heart attack victims Quickly degraded in blood by plasmin, also a protease Treatment involves infusion over long time period to dissolve clot Plasmin cleaves peptide bond after Lys or Arg Streptokinase Lys 59 and 386 changed to Gln by site directed mutagenesis Double mutant was 21 x more resistant to proteolysis by plasmin Could be administered sooner after heart attack by single injection in the field rather than as infusion after transport of patient to hospital
Produced by Streptococcus
Protease that dissolves blood clots, used to treat heart attack victims
Quickly degraded in blood by plasmin, also a protease
Treatment involves infusion over long time period to dissolve clot
Plasmin cleaves peptide bond after Lys or Arg
Streptokinase Lys 59 and 386 changed to Gln by site directed mutagenesis
Double mutant was 21 x more resistant to proteolysis by plasmin
Could be administered sooner after heart attack by single injection in the field rather than as infusion after transport of patient to hospital
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