Transcript Study and engineering of gene function: mutagenesis

Study and 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
Course Packet: #30
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
Animation of phage display
http://www.dyax.com/discovery/phagedisplay.html
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????