Expressing proteins from cloned genes continued

1
Expressing proteins from cloned genes continued
c. Expression using vectors in yeast
cells -advantages -single-celled eukaryotes
known to perform many posttranslational
modifications -easier and less expensive to work
with than insect or mammalian cells -ideally
suited for large-scale production in fermentation
apparatus -availability of yeast expression
plasmids that are shuttle vectors that can
replicate in both E. coli and yeast -one
commercially available system is the ESP2
system from Stratagene -this system allows
expression of GST/FLAG-tagged proteins in
Schizosaccaromyces pombe -having the GST fusion
allows easy column purification of the expressed
protein (just as with bacterially-expressed
GST-fusion proteins) -the FLAG tag is a strong
epitope tag, against which commercially available
antibodies are available -if desired the tags,
can be removed with thrombin or enterokinase
cleavage -three vectors are available, pESP-1,2
and 3, which vary in the restriction sites in
their MCS region that one can use to insert a
cDNA
2
(No Transcript)
3
-protein expression from the pESP vectors is
tightly controlled by the nmt1 (no message in
thiamine) promoter -the promoter is regulated by
the concentration of thiamine in the
media -induction of the promoter leads to a
300-fold increase in expression -the yeast
Pichia pastoris has emerged as a popular choice
for expression of cloned eukaryotic genes -P.
pastoris is a methylotrophic yeast which, in the
absence of a repressing carbon source such as
glucose, can utilize methanol as a carbon
source -the alcohol oxidase promoter (AOX1)
controls expression of alcohol oxidase, the
enzyme catalyzing the first step in the methanol
metabolic pathway - gt30 of the total soluble
protein in methanol-induced cells is alcohol
oxidase -Pichia expression vectors use the AOX1
promoter to drive high-level, tightly controlled
expression of recombinant proteins Some of the
advantages of using P. pastoris -extremely
high yields of intracellular proteins at levels
typically higher than in bacterial, insect, or
mammalian systems (grams per liter range) -high
levels of secretion into the medium -ease of
fermentation to very high cell density in simple
defined media -genetic stability and scale up
without loss of yield -Pichia expression kits
are marketed by Invitrogen
4
(No Transcript)
5
-pPIC vectors contain no ARS and are incorporated
into the host genome by homologous recombination
at the AOX1 locus -the antibiotic Zeocin can be
used to check for presence of vector in both
bacterial and yeast hosts -the vector is
generally introduced into two different kinds of
host Mut and Muts (methanol utilization
slow) - Mut cells have an intact AOX1 gene,
whereas Muts cells have a disrupted AOX1
gene -one host may express the protein of
interest more than the other, and both types
should be tested -higher levels of protein
expression can be achieved through multicopy
integration of the vector into the genome and a
number of tricks have been devised to select for
this -the pPIC9 vector contains a kanamycin
resistanec gene which conveys resistance to
Geneticin in Pichia and kanamycin in E.
coli -hosts with muliple incorporations of the
vector will grow in high concentrations of
Geneticin
6
(No Transcript)
7
(No Transcript)
8
-another technique uses an in vitro approach to
generating multicopy inserts -the gene of
interest is cloned into EcoR I site of the vector
pAO815 and then liberated as an expression
cassette by digestion with BamH I and Bgl
II -concatemers of the expression cassette are
generated by ligation in vitro, and multiple
copies are inserted back into the pAO815 vector
and transformed into Pichia
9
(No Transcript)
10
Transgenic animal approaches a. Use of
transgenes to study gene function in
non-vertebrate model organisms Transgenes in
Drosophila -the technology to make transgenic
Drosophila came from the molecular
characterization of P elements in the early
1980s -P elements encode a transposase that
enables them to insert into DNA -the gene
encoding the transposase in the P element makes
a functional protein when all three introns are
spliced out -normally, splicing of the third
intron only occurs in the germline, in somatic
tissues this intron is left in and contains a
stop codon -in somatic tissues a shorter,
nonfunctional transposase is made -a P element
(D 2-3) has been constructed that has the third
intron removed in the gene encoding the
transposase this allows transposase to be
produced in any tissue -the inverted repeats at
the ends of this element have been engineered
such that it itself cant transpose and this is
used as helper for transgene insertion
11
-the most commonly used approach to visualize
incorporation of a P element is to put in the
white (w) gene as a marker -the white gene is
required for red eye colour in the Drosophila
eye, by injecting the transgene-bearing P
element into w- embryos one can spot P element
incorporation by an orange to red eye
colour -the early vectors used to make
Drosophila transgenic lines allowed for
ubiquitous expression under control of promoters
such as that from the Actin5C gene -the
development of vectors containing the
promoter from the hsp70 gene allowed for temporal
control of transgene expression as this promoter
is only strongly active at high temperatures
such as 37oC -hsp70 transgenes can be kept
quiet at room temperature and induced by
shifting to 37oC -the most commonly used system
today is the GAL4-UAS system which allows one to
express transgenes in tissue specific
patterns -this vector takes advantage of a yeast
transcriptional activation system to control
expression of the transgene -in yeast, the
transcription factor GAL4 will trigger the
activation of genes bearing the upstream
activating sequence (UAS)
D2-3
12
-in pUAST, the gene of interest is cloned next to
five copies of UAS together with a
minimal promoter - series of fly strains have
been created that will express GAL4 either in
specific tissues or in response to heat
shock -thus, we can control the expression of
our transgene in various ways by mating to
different GAL4 strains
13
(No Transcript)
14
-two approaches have been taken in the
creation of GAL4 driver lines -one is to link a
cloned, characterized promoter upstream up GAL4
and introduce into flies -for example we could
use the hsp70 promoter which allows for heat
shock control of gene expression -the construct
for making such a transgene is assembled in
three way ligation between (i) the desired
promoter fragment (ii) the BamHI/NotI fragment
of pGaTB (iii) a P element vector such as
pCaSpeR -the other approach to creating GAL4
driver lines is through the technique of
enhancer trapping -in this technique, various
insertions of the vector pGawB in the genome
are isolated and tested for insertion of the GAL4
gene next to an endogenous enhancer that conveys
an interesting tissue-specific pattern of
expression -expression is tested by crossing the
GAL4 line to a reporter gene such as UAS-lacZ or
UAS-GFP and seeing where the reporter is
expressed
15
(No Transcript)
16
-heat shock expression of transgenes allows
precise temporal control, whereas use
of tissue-specific promoters/enhancers allows
for precise spatial control -the best situation
is obviously to be able to control both the
spatial and temporal pattern of transgene
expression -Halfon and colleagues have shown
that transgenes under control of a heat
shock promoter can be turned on in single
cells using a laser microbeam -a 1 to 2 minute
laser treatment can induce a heat shock but is
not lethal to the heat shocked cells -another
approach is to inject a caged light- inducible
form of a hybrid GAL4-VP16 transcription factor
into UAS-transgene embryos -GAL4-VP16 is caged
by reaction with 6- nitroveratrylchlorofromate
(NVOC-Cl) which blocks the ability of GAL4-VP16
to bind the UAS target sequence
17
-the ability of caged GAL4-VP16 to bind UAS and
activate transcription is restored by
illumination with 365-nm UV light -by aiming a
fine beam of light at a single cell, the caged
GAL4-VP16 will be photoactivated in that cell
alone and drive UAS-transgene expression in that
cell alone
18
-the two approaches just discussed are very
technically demanding -the recently described
GeneSwitch system is a more straightforward
technique for spatial and temporal regulation of
transgene expression -GeneSwitch makes use of a
GAL4- progesterone receptor fusion protein to
drive UAS-transgene expression -this
transcription factor is not functional unless
the drug RU486 is supplied -the spatial pattern
of GeneSwitch is supplied by a tissue-specific
enhancer promoter, while temporal specificity is
controlled by application of RU486
19
(No Transcript)