TITLE DATE
Protein Engineering
↳
modifying protein sequence by substitution or indels
Purposes -
probe mechanism (generate specific substitution, observe effect on structure & function (
-create novel proteins (w/ new functions and ..
applications (
e.g. improve catalytic function of enzyme
-alter substrate specificity or stereospecificity
-improve stability (stable enough for downstream applications (
-biotech applications 4: requirements 4
↳
e.g. more eco-friendly production
Mutagenesis
synthetic gene route: design substitution in specific locations
e.g. sub out of CyS
send to a
company like Thermo Fisher
time efficient
*
plasmid-based approach
e.g. Quikchange protocol from Stratagene (company (
1.
identify target site for mutation in parental
Plasmid DNA
2. high temperature to denature plasmid
lower temperature to anneal mutagenic
oligonucleotide primers
3.
Amplify DNA using PCR so new strand contains
mutation
add Dpn l enzyme
4. Wild
type (parental) DNA has restriction sites
by DpnI
cleares wild type, all remaining DNA is mutated
5. Transform mutated plasmid (nicked, dsDNA) into
XL1- Blue supercompetent cells and then E. coli
6. Sequencing repair nicks produce more
mutated DNA
↳ check for correct mutation, avoid introduction of random mutations
https://bynikkib.com
, TITLE DATE
Mutate genomic DNA (non-plasmid): overlap extension method using PCR
4 Oligonucleotide primers:
-forward and reverse primers
Introduces mutation
Mutagenesis (overlapping region
3: -upstream and downstream
5'
primers contain specific
5' 31
restriction enzyme sites
produce 2 PCR products
-
Oligo 104 (upstream + reverse
-
oligo 203(downstream+ forward)
denature and anneal in presence of
-full length pck product w/ both unique RE sites at oligo primers 102
3' and 5' ends and mutation at the middle
DNA polymerase makes as DNA
cut wild type and mutagenic DNA at 2 RE sites, replace WT w/ mutated and anneal
Alanine Scanning Mutagenesis
↳
systematic approach to engineer proteins (change a
region / whole protein one AA at a time
-used to investigate role of residues
-
obtain a more stable construct
mutate every amino acid residue into Ala one by one
Ala
*
is mutated into Lea
means of assessing the effect of mutations relative to wild type: normally Functional Assay
example: used to stability of G-protein coupled receptor (serrano-vega etal, 2008 (
systematic ... slow
https://bynikkib.com
Protein Engineering
↳
modifying protein sequence by substitution or indels
Purposes -
probe mechanism (generate specific substitution, observe effect on structure & function (
-create novel proteins (w/ new functions and ..
applications (
e.g. improve catalytic function of enzyme
-alter substrate specificity or stereospecificity
-improve stability (stable enough for downstream applications (
-biotech applications 4: requirements 4
↳
e.g. more eco-friendly production
Mutagenesis
synthetic gene route: design substitution in specific locations
e.g. sub out of CyS
send to a
company like Thermo Fisher
time efficient
*
plasmid-based approach
e.g. Quikchange protocol from Stratagene (company (
1.
identify target site for mutation in parental
Plasmid DNA
2. high temperature to denature plasmid
lower temperature to anneal mutagenic
oligonucleotide primers
3.
Amplify DNA using PCR so new strand contains
mutation
add Dpn l enzyme
4. Wild
type (parental) DNA has restriction sites
by DpnI
cleares wild type, all remaining DNA is mutated
5. Transform mutated plasmid (nicked, dsDNA) into
XL1- Blue supercompetent cells and then E. coli
6. Sequencing repair nicks produce more
mutated DNA
↳ check for correct mutation, avoid introduction of random mutations
https://bynikkib.com
, TITLE DATE
Mutate genomic DNA (non-plasmid): overlap extension method using PCR
4 Oligonucleotide primers:
-forward and reverse primers
Introduces mutation
Mutagenesis (overlapping region
3: -upstream and downstream
5'
primers contain specific
5' 31
restriction enzyme sites
produce 2 PCR products
-
Oligo 104 (upstream + reverse
-
oligo 203(downstream+ forward)
denature and anneal in presence of
-full length pck product w/ both unique RE sites at oligo primers 102
3' and 5' ends and mutation at the middle
DNA polymerase makes as DNA
cut wild type and mutagenic DNA at 2 RE sites, replace WT w/ mutated and anneal
Alanine Scanning Mutagenesis
↳
systematic approach to engineer proteins (change a
region / whole protein one AA at a time
-used to investigate role of residues
-
obtain a more stable construct
mutate every amino acid residue into Ala one by one
Ala
*
is mutated into Lea
means of assessing the effect of mutations relative to wild type: normally Functional Assay
example: used to stability of G-protein coupled receptor (serrano-vega etal, 2008 (
systematic ... slow
https://bynikkib.com
