Study and engineering of gene function: mutagenesis

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12 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Study and engineering of gene
function: mutagenesis

I.
Why mutagenize?

II.
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

From Koeller and Wang, “enzymes for chemical synthesis”, Nature 409, 232
-

240 (2001)

Obtaining useful enzymes

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 Mn
2+

in reaction


Reduce concentration of one dNTP

Random mutagenesis by
PCR: the Green Fluorescent
Protein

Screen mutants

Cassette mutagenesis
(semi
-
random)

Strands synthesized individually, then annealed


Allows random insertion of any amino acid at defined positions

Translation of sequence

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: 10
9

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

Human growth hormone: want to generate variants that
bind to hGH receptor more tightly

Random mutagenesis

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

“QuikChange
TM
” protocol

Advantage: can use plasmid (double
-
stranded) DNA

Destroys the
template DNA
(DNA has to
come from
dam
+

host

Site
-
directed
mutagenesis:
Mega
-
primer
method

Megaprimer needs to be
purified prior to PCR 2


Allows placement of
mutation anywhere in a
piece of DNA

A

B

Wild type template

First PCR

Second PCR

Domain swapping using “megaprimers” (overlapping PCR)

N
-

-
C

Mega
-
primer

PCR 1

PCR 2

Domains have been swapped

Template 1

Template 2

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 tRNA
tyr
, and the tRNA
tyr

synthetase
(mTyrRS) from an archaean (M.jannaschii)
--
no
cross
-
reactivity with E. coli tRNA
tyr

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
-
L
-
phenylalanine

m
-
acetyl
-
L
-
phenylalanine

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????