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Optimization of the Bacterial Expression System for LIN-12/Notch-Repeats
(LNRs) from Human Notch1 and 2
Ursela Siddiqui
Advisor: Dr. Didem Vardar-Ulu
Wellesley College
Protein expression is the translation and post-translational processing of proteins. The ability to control and
manipulate this process to obtain only a desired product of a target gene is essential for biochemical research.
There are four commonly used expression systems: mammalian, insect, yeast and bacteria. Due to its simplicity
and high yields, biochemists heavily rely on an Escherichia coli based bacterial expression system to obtain their
protein of interest for further studies. On the other hand, many parameters need to be first optimized to fully exploit
this system for a particular target protein. Additionally, the protein of interest can be either soluble or insoluble
under the specific expression conditions and there are advantages and disadvantages to both approaches. The
first part of this project focuses on the optimization of bacterial expression for the first two tandem Lin12/Notch
Repeats, LNRA and LNRB (LNRAB) from Human Notch1 and 2. Here, we report a comprehensive analysis of the
optimization findings and the determined optimal set of expression conditions. The second part of the project is
aimed to combine different components of the soluble and insoluble expression systems into an innovative
expression and purification methodology in an attempt to alleviate some of the disadvantages each system has
separately. Here, we report the preliminary results on the initial steps of this hybrid methodology and outline the
remaining steps with the expected outcomes.
The two major components of any bacterial expression system are:
1. A plasmid vector: a small circular molecule of double stranded DNA derived from natural plasmids that occur in
bacterial cells with a piece of inserted DNA that codes for the desired protein.
2. A bacterial cell line with the desired growth and expression characteristics. Bacterial cell lines differ in the
components they bring with them to the expression system. The most widely used hosts for protein expression are the BL21
strain which are
deficient in both lon and ompT proteases. The cells used in the current study are all the DE3
derivatives of the BL21 line, which allows for induction by IPTG because it renders the cells under the control of the lac
promoter.
Table 1. Optimization Parameters and Results
Cell
Time1
IPTG concentration2
PlysS
initial gradual
increase, then
saturation at 3 hrs
No difference when testing
from 0.1 to 0.5 mM
Origami
No difference
No difference
0.7 OD showed a
darker band than
0.5 OD
OrigamiPlysS
initial gradual
increase, then
saturation at 3 hrs
No difference
0.7 OD showed a
darker band than
0.5 OD
Rosetta-gamiPlyss
initial gradual
increase, then
saturation at 3 hrs
No difference
0.7 OD showed a
darker band than
0.5 OD
RIPL
No difference
No difference
inconclusive
RosettaPlysS
initial gradual
increase, then
saturation at 3 hrs
Subtle difference showing
darker bands at 0.3 mM versus
0.1 mM
0.8 OD showed a
darker band than
0.6 OD
1)
2)
3)
4)
Cell Density3
Growth Media1
No difference
Analysis of samples representing the different optimization parameters by SDS-PAGE show that regardless of
cell line, much lower concentrations of IPTG than the typical values used in literature (0.5mM) and shorter
induction times can be used in the expression of the tested constructs without any significant loss in production
yield. We have also noted that even for 4hrs of expression, there is not a significant amount of protein
degradation. Therefore, an IPTG concentration of 0.25 mM and induction time of 4 hours will be used for future
large scale expressions. Furthermore, on those cell lines tested, a higher cell density does produce a more
noticeable band on the SDS-PAGE. A density of 0.7 OD600 will be used for a larger scaled growth. For the cells
being discussed currently, growth media does not seem to impact protein production. LB Broth Miller will be
used in any future studies as the growth media.
Project 2: Development of a new hybrid expression and purification protocol.
Single large scale growth and expression under optimized conditions followed by purification
.
Add appropriate solution,
centrifuge and collect
supernatant
Induced samples were taken at time points: 1hr, 2hrs, 3hrs, and 4 hrs post induction.
IPTG concentrations tested ranged for 0.01, 0.05, 0.1, 0.2, 0.3,0.4, and 0.5mM.
Cell density as measured at 600 nm by a spectromoter ranged from 0.5 OD to 0.9 OD
Growth media used were LB Broth Miller and LB Broth Lennox. LB Broth Miller Contains 10g/L salt whereas LB Broth
Lennox has 5 g/L
WASH 1: 10 mM immidazole
WASH 2: 10 mM immidazole
Further subtypes used in the current study are:
1. PlysS strains that express T7 lysozyme which stabilizes growth prior to IPTG addition.
2. Rosetta cells that have the tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA. These are codons that are typically
used by the mammalian systems to code for amino acids and hence E. coli normally do not contain tRNAs to translate
these codons. These cell lines allow for more efficient translation of mammalian genes in the otherwise limited
expression system.
3. Origami cells that have mutations which allow them to support disulfide bond formation (normally E. coli cellular
environment is too reducing for this post translational modification to take place)
4. Rosetta-gami cells that have the characteristics of both the Rosetta and Origami cell lines.
There are three major steps in any bacterial expression system. The first step is transformation during which the cells
take up the vector. The second is growth and protein expression, where the bacteria are allowed to grow to a certain cell
density and then stimulated to begin the expression of the protein. The third is isolation and purification where the desired
protein is separated and collected. It is the specifics of the last two steps that differentiate one expression system from
another based on the solubility of the expressed protein in the employed system. In general insoluble systems result in
higher protein expression levels however there is a need to employ harsh conditions to solubilize the desired protein during
purification. On the other hand, gentler purification conditions for soluble proteins make them also more prone for
degradation, especially if they are small.
REPRESENTATIVE DATA
MW Marker
1hr
2hr
3hr
4hr
500 ml growth using
ideal parameters
found from project 1
Figure 2: The effect of time on protein production. The band
indicated by the arrow is the protein of interest seems to reach
full saturation at three hours showing subtle differences in band
darkness between one and two hours. Time was tested for a
total of four hours. The vector and cell line used in this growth was
250
150
100
75
50
37
Expected
MW:
17033.3
15
10
Since each LNR is ~35 residues in length, they are extremely susceptible to degradation if produced as soluble
proteins in E.coli. To thwart this problem, we decided to express a larger version of the protein initially, namely LNRAB
using the pET15b vector based His-tagged soluble protein expression system and then modify it through a specific
cleavage normally used during the purification steps of an insoluble expression protocol, to obtain the two individual LNRs
as the desired pieces. This new hybrid strategy exploits the power of affinity chromatography for purifying the His-tagged
target protein using Nickel beads. After the purification and the in vitro folding of the expressed protein, this new protocol
takes advantage of a naturally occurring methionine residue between LNRA and LNRB in human Notch2 sequence to
separate the polypeptide into the two individual LNRs through cleavage with cyanogen bromide. Since the individual LNRs
have different physicochemical properties, after cleavage they can be separated using a reverse-phase C18 HPLC.
Figure 7: His-tag purification of RosettaPlysS cells
from human Notch2 LNRAB. Multiple bands
indicate the purification was not complete and other
components are still present. Most importantly, the
multiple bands in E1 and E2 indicate this.
Day long growth and expression:
50 ml LB Broth Miller
50 µl 1000X vector/cell-line specific antibiotic
ampicillin
kanamycin
chloromphenicol
0.5 ml of an overnight culture
.
0.2mM 0.3mM 0.4mM 0.5mM
250
150
100
75
50
37
25
Notch2 LNR_AB in pET15b:
20
Expected
MW:
17033.3
kD
0.7 OD600
1hr 2hr 3hr 4hr
Figure 4: The effect of cell density measured at 600nm
on protein production. The band indicated by the arrow
is the protein of interest and is darker for the higher cell
density at induction. This experiment was conducted
with Origami cells containing pET32a carrying human
Notch1 LNRAB DNA.
250
150
100
75
50
Incubate
overnight
at 37 °C
25
20
15
Figure 1:Procedure for testing parameters used in bacterial expression. After adding LB Broth Miller, the appropriate
antibiotics and the vector+cell lines, the solution incubates overnight at 37 C. The next day 0.5 ml of this
culture is added to a multiple new flasks containing fresh LB Broth Miller and the appropriate antibiotics. Cell
growth is monitored by checking samples for absorbance at 600nm and then inducing them by the addition of
IPTG. Finally, hourly samples are collected after IPTG addition and run on an SDS PAGE under reducing
conditions.
Expected
MW:
31513.0
25
20
15
kD
TEV
Recognition Site
Used to cleave off
the affinity tag
from the protein
construct
LNR- A
Met
LNR- B
Used to
separate
LNRA from
LNRB
Figure 8: Different elements of human Notch2 LNRAB construct used during the hybrid new purification
protocol to obtain individual LNRs (LNR-A and LNR-B)
Expected
MW:
26866.8
Unind. 1 hr
Run samples on SDS-PAGE gel
37
37
LB Broth Miller
Take hourly 1 ml sample
from each flask after
induction with desired
concentration of IPTG
250
150
100
75
50
Met G S S H H H H H H S S G L V P R G S H Met E N L Y F Q G A T C L S Q Y C A D K A R D G V C D E A C N
S H A C Q W D G G D C S L T Met E N P W A N C S S P L P C W D Y I N N Q C D E L C N T V E C L F D N F E
C Q Stop
Affinity tag
already on
vector
Preliminary results from purification steps seem to indicate we were able to get enough hN2LNRAB eluted off of
Nickel beads to carry out the rest of the proposed protocol. Future directions will involve evaluating the success
of the new methodology by using a larger construct consisting of human Notch2 NRR. In addition, cleavage
reagents such as cyanogen bromide will be used to attempt to separate constructs grown together. Even though
we expressed and partially purified the target protein, our gel indicates that there are at least two significant
species in the eluted fraction, so first we need to identify them and purify the desired one, then perform an in vitro
folding to obtain the correct disulfide bonds and then cleave the polypeptide into two to obtain the individual
LNRs.
10
kD
Add
*Lysis Buffer:
50 mM Tris H-Cl, pH 8.0
200 mM NaCl
20 mM Imidazole, pH 8.0
NOTE: Wah and Elution buufers only differ
in immadazole concentration
10
His Tag
0.5 OD600
MW
Marker Unind 1 hr 2hr 3hr 4hr
Project 1: Optimization of Bacterial Expression
Multiple simultaneous small scale growths to test for various expression parameters:
Overnight cultures:
5 ml LB Broth Miller
5 µl 1000X vector/cell-line specific antibiotic
ampicillin
kanamycin
chloromphenicol
Scoop of a glycerol stock or a freshly
transformed colony
MW marker 0.1mM
Figure 3: The effect of IPTG concentration on protein
production. The band indicated by the arrow is the
protein of interest and does not get significantly darker
as the concentration of IPTG increases from 0.1mM to
0.5mM. Concentrations not shown that were also
tested are 0.01mM and 0.05 mM. These did not show
difference either. The vector and cell line used in this
growth was pMML vector containing Notch1 LNR-A
DNA in PlysS cells.
ELUTION 2: 500 mM immidazole
MW unind ind SUP1 SUP2 FT W1 W2 E1 E2
kD
For the first part of the project we have used several combinations of vectors and cell-lines to systematically vary
several parameters within the expression system to obtain the maximum protein yield and monitored their impact on protein
production via SDS-PAGE analysis. The main variables that were assayed during this study were different cell densities at
the time of IPTG addition (induction), IPTG concentrations used for induction, total expression time, as well as growth
media.
ELUTION 1: 300 mM immidazole
Add supernatants
to Nickel beads,
incubate at 4 C for
1 hour, centrifuge
and collect
supernatant (FT)
Figure 6: Steps for purification after growth and protein expression. In order to lyse the cells, a sonicator is used.
This solution is then centrifuged and the supernatant is collected and added to approximately 250 ml of preequilibrated Nickel beads. The His-tag on the protein has an affinity for these beads. To elute the protein off the
Nickel beads, a buffer containing high immidazole concentration is used. Immidazole competes for the same
binding site on the Nickel beads as the histidines.
pMML vector containing Notch1 LNR-A DNA in PlysS cells.
25
20
Centrifuge remainder
and resuspend pellet
in Lysis buffer*,
sonicate and
centrifuge. Repeat
(SUP1, SUP2)
Figure 5: The effect of growth media on protein
production. The band indicated by the arrow is
the protein of interest and does not differ in
darkness from one growth media to another. The
difference in growth media is LB Broth Miller has
10 g/L salt concentration whereas LB Broth
Lennox has 5 g/L. The vector and cell line used
in this growth was pMML vector containing
Notch1 LNR-A DNA in PlysS cells.
2hr
LB Broth Lennox
3hr
4hr
1hr
2hr
3hr
4hr
Unind
Expected
MW:
17033.3
Dr. Didem Vardar-Ulu
Wellesley College
The Roberta Day Staley and Karl A. Staley Fund for Cancer-Related Research
Vardar Ulu Lab
Christina Hao
Fahmi Jahufar
Sharline Madera