Recombinant engineering of
gene function: mutagenesis
I.
II.
Why mutagenize?
Random mutagenesis, mutant
selection schemes
III. Site-directed mutagenesis,
deletion mutagenesis
IV. Engineering of proteins
V. Alterations in the genetic code
Lecture for Bio FMIPA UB, by Fatchiyah
Uses for mutagenesis
• Define the role of a gene--are phenotypes
altered by mutations?
• Determine functionally important regions of a
gene (in vivo or in vitro)
• Improve or change the function of a gene
product
• Investigate functions of non-genes, eg. DNA
regions important for regulation
Protein engineering-Why?
• Enhance stability/function under new conditions
– temperature, pH, organic/aqueous solvent,
[salt]
• Alter enzyme substrate specificity
• Enhance enzymatic rate
• Alter epitope binding properties
Enzymes: Biotech Cash Crops
Obtaining useful enzymes
From Koeller and Wang, “enzymes for chemical synthesis”, Nature 409, 232 - 240 (2001)
Random mutagenesis
• Cassette mutagenesis with “doped”oligos
• Chemical mutagenesis
– expose short piece of DNA to mutagen, make
“library” of clones, test for phenotypes
• PCR mutagenesis by base
misincorporation
– Include Mn2+ in reaction
– Reduce concentration of one dNTP
Random mutagenesis by
PCR: the Green Fluorescent
Protein
Screen mutants
Cassette mutagenesis
(semi-random)
Translation of sequence
Strands synthesized individually, then annealed
Allows random insertion of any amino acid at defined positions
Random and semi-random mutagenesis:
directed evolution
• Mutagenize existing protein, eg. error-prone PCR,
doped oligo cassette mutagenesis
-- and/or -Do “gene shuffling”
(Creates Library)
• Screen library of mutations for proteins with altered
properties
– Standard screening: 10,000 - 100,000 mutants
– Phage display: 109 mutants
Gene shuffling: “sexual PCR”
Gene shuffling
For gene shuffling
protocols you must have
related genes in original
pool: 1) evolutionary
variants, or 2) variants
mutated in vitro
Shuffling allows rapid
scanning through
sequence space:
faster than doing
multiple rounds of
random mutagenesis
and screening
Shuffling of one gene mutagenized in two ways
Gene shuffling--cephalosporinase from 4 bacteria
Single gene mutagenesis
Multiple gene shuffling
Screening by phage display: create library of
mutant proteins fused to M13 gene III
Random mutagenesis
Human growth hormone: want to generate variants that
bind to hGH receptor more tightly
Phage display:production of recombinant phage
The “display”
Phage display: collect tight-binding phage
The selection
Types of Mutagenesis
• PCR Based Methods
–
–
–
–
Site-directed mutagenesis
Mismatched mutagenesis
5’ add on mutagenesis
Cassette Mutagenesis
• Insertional Mutagenesis
– Trasposon mutagenesis
• In vivo Mutagenesis
– Direct Mutagenesis
Mismatched Mutagenesis
• Similar to Site-Directed Mutagenesis
• But only focuses on a single amino acid
• Important when trying to determine a
particular missense mutation in known gene
of a disease.
• Or when just trying to evaluate the
contributation of the single amino acid to
the function of the protein.
Gene or cDNA
is cloned into
M13 vector
Use of M13 allows
for single strand
recombinant DNA
recovery
Screen for
mutant
5’ add on Mutagenesis
• Involves adding on a new sequence or
chemical group to the 5’end of a PCR
product
• This involves a particular way of designing
the primers:
– 3’ end of the primer matches the sequence of
PCR product.
– 5’ end contains the novel sequence
• Suitable restriction site
• Addition of a functional sequence (promoter
sequence)
Uses and Limitations
• PCR based methods are
useful in making specific
mutations in the DNA
• Which is useful when
studying different aspects
of protein function
• With PCR based methods
it is hard to replicate the
mutated DNA…in order
for replication to occur
super competent cells
must be used and are
expensive!
• Screening can be tedious,
usually requires
sequencing to confirm if
mutation occurred.
Cassette Mutagenesis
• Used to introduce multiple mutations into
the DNA sequence
• Uses blunt ended DNA for insertion site of
mutation
• Where mutation is inserted a 3 base pair
direct terminal repeat is created
• The mutagenic codon cassette has two head
to head SapI sites allowing for removal of
all DNA except for mutation.
Targeted codon
removed using
restriction enzyme
that creates blunt
cut
SapI digestion
creates 3’
overhang allowing
for ligation.
Ligation, which
creates final mutation
Uses and Limitations
• Typically used for
protein structure but
possibly used for gene
function
• Less expensive than site
directed mutagenesis to
create several mutations,
because there is no need
for primers
• Requires the SapI
restriction enzyme cut
sites, and other cut sites
flanking the target
region for removal of
DNA
• Works best when target
region is contained in a
small DNA fragment
Transposon Mutagenesis
• Transposon: a piece of short DNA that replicates by inserting into
other pieces of DNA (plasmids, chromosomes, etc…)
• Useful for studying gene function because when the transposon
moves into different location in the DNA it may cause a disruption
in a gene or a set of genes.
• Transposons also have many useful properties for mutagenesis:
– Cause clean mutations
– Can be random or specific mutations
– Typically encode for antibiotic resistance or some other
advantageous gene.
– Can use a transposon that inserts at a high frequency
– When used in bacteria it causes selectable phenotypes
– Recognize specific sequence that is ~2-12 base pairs long
Uses and Limitations
• Primary use is for the study
of gene function, though can
be used to create gene
fusions
• Usually easy to see a change
in phenotype due to gene
knockout
• Because the transposon
inserts at a specific
sequence, helps in
determining where insertion
occurred
• Not useful in large plasmids
because many recognition
sites could be contained in
the a single plasmid
• Suicide vectors are used,
though some may have
limited replication, so further
screening is needed
Site-directed
mutagenesis: primer
extension method
Drawbacks:
-- both mutant and wild type
versions of the gene are made
following transfection--lots of
screening required, or tricks
required to prevent replication of
wild type strand
-- requires single-stranded,
circular template DNA
Alternative primer extension
mutagenesis techniques
TM
“QuikChange ”
protocol
Destroys the
template DNA
(DNA has to
come from
dam+ host
Advantage: can use plasmid (double-stranded) DNA
Site-directed
mutagenesis:
Mega-primer
method
First PCR
A
Second PCR
Wild type template
B
Megaprimer needs to be
purified prior to PCR 2
Allows placement of
mutation anywhere in a
piece of DNA
Domain swapping using “megaprimers” (overlapping PCR)
-C
N-
Template 1
PCR 1
Mega-primer
Template 2
PCR 2
Domains have been swapped
PCR-mediated deletion mutagenesis
Target DNA
PCR products
Oligonucleotide design allows precision in deletion positions
Directed mutagenesis
• Make changes in amino acid sequence
based on rational decisions
• Structure known? Mutate amino acids in
any part of protein thought to influence
activity/stability/solubility etc.
• Protein with multiple family members?
Mutate desired protein in positions that
bring it closer to another family member
with desired properties
An example of directed
mutagenesis
T4 lysozyme: structure known
Can it be made more stable by
the addition of pairs of cysteine
residues (allowing disulfide
bridges to form?) without altering
activity of the protein?
T4 lysozyme: a model for stability studies
Cysteines were added to
areas of the protein in
close proximity--disulfide
bridges could form
More disulfides, greater stabilization at high T
Bottom of bar:
melting temperature
under reducing
condtions
Top of bar:
Melting temperature
under oxidizing
conditions
Green bars: if the
effects of individual
S-S bonds were
added together
Stability can be increased - but there can be a cost in activity
The genetic code
• 61 sense codons, 3 non-sense (stop) codons
• 20 amino acids
• Other amino acids, some in the cell (as precursors to other
amino acids), but very rarely have any been added to the
genetic code in a living system
• Is it possible to add new amino acids to the code?
• Yes...sort of
Wang et al. (2001) “Expanding the genetic code” Science 292,
p. 498.
Altering the genetic code
Why add new amino acids to proteins?
• New amino acid = new functional group
• Alter or enhance protein function (rational
design)
• Chemically modify protein following synthesis
(chemical derivitization)
– Probe protein structure, function
– Modify protein in vivo, add labels and
monitor protein localization, movement,
dynamics in living cells
How to modify genetic code?
Adding new amino acids to the code--must bypass the
fidelity mechanisms that have evolved to prevent this
from occurring
2 key mechanisms of fidelity
•
Correct amino acid inserted by ribosome through
interactions between tRNA anti-codon and mRNA codon
of the mRNA in the ribosome
•
Specific tRNA charged with correct amino acid because
of high specificity of tRNA synthetase interaction
•
Add new tRNA, add new tRNA synthetase
tRNA charging and usage
Charging:
(tRNA + amino acid + amino
acyl-tRNA synthetase)
Translation:
(tRNA-aa +
codon/anticodon
interaction + ribosome)
• Chose tRNAtyr, and the tRNAtyr synthetase
(mTyrRS) from an archaean (M.jannaschii)--no
cross-reactivity with E. coli tRNAtyr and synthetase
• Mutate m-tRNAtyr to recognize stop codon (UAG)
on mRNA
• Mutate m-TyrRS at 5 positions near the tyrosine
binding site by doped oligonucleotide random
mutagenesis
• Obtain mutants that can insert O-methyl-L-tyrosine
at any UAG codon
Outcome
• Strategy allows site specific insertion of new
amino acid--just design protein to have UAG stop
codon where you’d like the new amino acid to go
• Transform engineered E. coli with plasmid
containing the engineered gene
• Feed cells O-methyl tyrosine to get synthesis of
full length gene
Utility of strategy
• Several new amino acids have been added to the E. coli
code in this way, including phenyalanine derivatives with
keto groups, which can be modified by hydrazide-containing
fluorescent dyes in vivo
– Useful for tracking protein localization, movement, and
dynamics in the cell
p-acetyl-Lphenylalanine
m-acetyl-Lphenylalanine
Some questions:
• What are the consequences for the cell with an
expanded code?
• Do new amino acids confer any kind of
evolutionary advantage to organisms that have
them? (assuming they get a ready supply of the
new amino acid…)
• Why do cells have/need 3 stop codons????