INTRODUCTION - scitech.technion.ac.il · PHASE #2 –OPTIMIZATION OF THE EXPRESSION Protein gel...
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What is the optimal way to purify and express a new BYK protein? Different methods from the DNA level to protein expression and purification to achieve an extremely pure protein for X-ray crystallography INTRODUCTION REFERENCES Antibiotics, drugs used to treat bacterial illnesses, are quickly becoming ineffective due to the rise of antibiotic resistant bacteria 1 . In the process of killing bacteria, these drugs put life or death pressure on the organisms, which most likely causes them to divide rapidly and mutate in an effort to survive, causing resistant strains that are harder to combat. The new goal is to make drugs that do not put the same pressure on the cells by harming their virulence without killing them. Thus, BY-kinases emerge as a potential target. BY-Kinases are a group of proteins that participate in many different functions, most notably in biofilm production by contributing exopolysaccharides 2 . Within bacteria, biofilm (an extracellular matrix) acts as a defence mechanism against drugs that may harm it 3 . The working hypothesis is that by inhibiting the expression of BY-Kinases and thus the contribution of exopolysaccharides, the biofilm is severely compromised, which may allow antibiotics to be effective in resistant bacteria. Therefore, the study of BY-kinase structure could be used to develop a new group of antibiotics that would aim to inhibit BY-kinase synthesis. The goal of this research is to compare methods of purifying and expressing BY-Kinase proteins in order to study their structure for further research as each protein is unique and requires optimization to the expression and purification protocol. Students: Juliana Martes Sternlicht, Tahlia Aviva Altgold Mentor: Michal Mayer, MSc candidate Assistant Prof. Meytal Landau Faculty of Biology, Technion [1] GRANGEASSE, Christophe; TERREUX, Raphaël; NESSLER, Sylvie. Bacterial tyrosine-kinases: Structure–function analysis and therapeutic potential. Biochimica Et Biophysica Acta (bba): Proteins and Proteomics, [s.l.], v. 1804, n. 3, p.628-634, mar. 2010. [2] MARCUZZO, Alberto Vito et al. Hyaluronate effect on bacterial biofilm in ENT district infections: a review. Apmis, [s.l.], 24 jul. 2017. Wiley- Blackwell. [3] HØIBY, Niels et al. Antibiotic resistance of bacterial biofilms. International Journal Of Antimicrobial Agents, [s.l.], v. 35, n. 4, p.322-332, abr. 2010. 2017 ACKNOWLEDGEMENTS We would like to thank Michal Mayer, MSc candidate, and Assistant Prof. Meytal Landau for hosting and guiding us through our research in her laboratory. We would also like to thank the TCSB for allowing us to use their facilities. Finally, we would like to thank the foundations and donors for their generous support of the SciTech Program. CONCLUSIONS During phase 1 (Figure 1), we digested wzc in pET9d, bceF in pET9d, and pET11d using restriction enzymes. As shown in Figure 4, the digestion was not completely successful for wzc and pET9d as well as pET11d. Although bceF was successfully digested, it was not successfully ligated unlike wzc, as seen in Figure 5 and Table 1. In phase 2 (Figure 2) the optimal expression conditions were found to be inducible expression (pET11d) at 37°C as seen in Figure 6. In the purification phase (Figure 3) we used a His-Trap affinity column (Figure 7) and a Size Exclusion Column (Figure 8) and achieved a large amount of protein, yet not sufficiently pure (there is no single band of Wzc, bands of smaller proteins appear as well). From Figure 8 that Wzc has a tendency to aggregate which affects its ability to crystallize. MATERIALS AND METHODS RESULTS Affinity column His-Trap column Cell membrane lysis French press Wzc expressed from pET11d vector induced with 1mM IPTG at 37°C PHASE #3 – PURIFICATION OF Wzc Size exclusion column Figure 1: Flowchart of methods of Phase #1 – Subcloning of bceF and wzc into pET11d Cell membrane lysis Sonicator Protein expression pET9d 37°C pET11d 37°C 1mM IPTG pET11d 16°C 0.1mM IPTG Transformation to BL-21 wzc in pET9d wzc in pET11d PHASE #2 – OPTIMIZATION OF THE EXPRESSION Protein gel Figure 2: Flowchart of methods of Phase #2 – Optimization of the Expression Figure 3: Flowchart of methods of Phase #3 – Purification of Wzc Figure 4: Gel results of the vector exchange of bceF and wzc from pET9d into pET11d. All samples were cut with BamHI-HF and NcoI-HF and ran in 1% agarose gel. Figure 5: Colony PCR gel results of the ligation of wzc into pET11d. C1 refers to colony 1 etc. CTL 1, 2, and 3 stand for, respectively, pET11d in XL1, wzc in pET9d, and no bacteria cells. All samples ran in 1% agarose gel. SAMPLE N° OF COLONIES WZC in pET11d in XL-1 (CIP) (regular concentration) 10 WZC in pET11d in XL-1 (CIP) (concentrated) 46 WZC in pET11d in XL-1 (CIP) (diluted) 1 Control 1 - WZC + pET11d in XL-1 (without ligase) (CIP) 62 Control 2 - WZC + pET11d in XL-1 (without ligase and insert) (CIP) 46 XL-1 colonies Table 1: Number of colonies in different concentrations of wzc an bceF in pET11d transformed in XL-1. Transformation E. Coli strain XL-1 Ligation into pET11d T4 DNA Ligase Digestion via restriction enzymes CIP PHASE #1 – SUBCLONING OF wzc AND bceF INTO pET11d Figure 7: Affinity column gel results of Wzc with coomassie stain on SDS-PAGE 12% acrylamide Figure 8: Size column gel results of Wzc with coomassie stain on SDS-PAGE 12% acrylamide PHASE #4 – CRYSTALLIZATION Figure 6: Gel results of Wzc in pET9d and pET11d with coomassie stain (A) on SDS-PAGE 12% acrylamide and Western-blot results (B) of same gel with antibody anti-His-HRP (Thermo) 1:1000 100bp marker 1 Kb marker 10,000 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750 3,000 1,500 1,000 900 800 700 600 500 400 300 500 250 200 wzc + pET9d pET11d bceF +pET9d pET11d 100bp marker 1 Kb marker 10,000 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750 3,000 1,500 1,000 900 800 700 600 500 400 300 500 250 200 C1 C2 C3 C4 C5 C1 C2 C3 CTL 1 CTL 2 CTL 3 Wzc in pET11d in XL1 (CIP) concentrated Wzc in pET11d in XL1 (CIP) regular wzc pET11d mw (kDa) ~25 ~35 ~48 ~63 ~75 kDa Wzc pET9d SUP 37C pET9d pellet 37C pET11d SUP 16C 0hr pET11d pellet 16C 0hr pET11d SUP 16C 20hr pET11d pellet 16C 20hr pET11d SUP 37C 0hr pET11d pellet 37C 0hr pET11d SUP 37C 1hr pET11d pellet 37C 1hr pET11d SUP 37C 2hr pET11d pellet 37C 2hr pET11d SUP 37C 4hr pET11d pellet 37C 4hr A mw (kDa) ~25 ~35 ~48 ~63 ~75 kDa Wzc pET9d SUP 37C pET9d pellet 37C pET11d SUP 16C 0hr pET11d pellet 16C 0hr pET11d SUP 16C 20hr pET11d pellet 16C 20hr pET11d SUP 37C 0hr pET11d pellet 37C 0hr pET11d SUP 37C 1hr pET11d pellet 37C 1hr pET11d SUP 37C 2hr pET11d pellet 37C 2hr pET11d SUP 37C 4hr pET11d pellet 37C 4hr B A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 Fractions ~25 ~35 ~48 ~63 ~75 kDa mw (kDa) ~25 ~35 ~48 ~63 C1 A5 A6 A7 A11 B3 B8 B9 B10 B12 Fractions A12 B1 B2 B11 ~75 kDa Wzc Wzc
Transcript of INTRODUCTION - scitech.technion.ac.il · PHASE #2 –OPTIMIZATION OF THE EXPRESSION Protein gel...
What is the optimal way to purify and express a new BYK protein?
Different methods from the DNA level to protein expression and
purification to achieve an extremely pure protein for X-ray crystallography
INTRODUCTION
REFERENCES
Antibiotics, drugs used to treat bacterial illnesses, are quickly becoming ineffective due to the rise of antibiotic resistant bacteria1. In the
process of killing bacteria, these drugs put life or death pressure on the organisms, which most likely causes them to divide rapidly and mutate
in an effort to survive, causing resistant strains that are harder to combat. The new goal is to make drugs that do not put the same pressure on
the cells by harming their virulence without killing them. Thus, BY-kinases emerge as a potential target. BY-Kinases are a group of proteins
that participate in many different functions, most notably in biofilm production by contributing exopolysaccharides2. Within bacteria, biofilm (an
extracellular matrix) acts as a defence mechanism against drugs that may harm it3. The working hypothesis is that by inhibiting the expression
of BY-Kinases and thus the contribution of exopolysaccharides, the biofilm is severely compromised, which may allow antibiotics to be effective
in resistant bacteria. Therefore, the study of BY-kinase structure could be used to develop a new group of antibiotics that would aim to inhibit
BY-kinase synthesis. The goal of this research is to compare methods of purifying and expressing BY-Kinase proteins in order to study their
structure for further research as each protein is unique and requires optimization to the expression and purification protocol.
Students: Juliana Martes Sternlicht, Tahlia Aviva Altgold
Mentor: Michal Mayer, MSc candidate Assistant Prof. Meytal Landau Faculty of Biology, Technion
[1] GRANGEASSE, Christophe; TERREUX, Raphaël; NESSLER, Sylvie. Bacterial tyrosine-kinases: Structure–function analysis and therapeutic
potential. Biochimica Et Biophysica Acta (bba): Proteins and Proteomics, [s.l.], v. 1804, n. 3, p.628-634, mar. 2010.
[2] MARCUZZO, Alberto Vito et al. Hyaluronate effect on bacterial biofilm in ENT district infections: a review. Apmis, [s.l.], 24 jul. 2017. Wiley-
Blackwell.
[3] HØIBY, Niels et al. Antibiotic resistance of bacterial biofilms. International Journal Of Antimicrobial Agents, [s.l.], v. 35, n. 4, p.322-332, abr. 2010.
2017
ACKNOWLEDGEMENTSWe would like to thank Michal Mayer, MSc candidate, and Assistant Prof. Meytal
Landau for hosting and guiding us through our research in her laboratory.
We would also like to thank the TCSB for allowing us to use their facilities.
Finally, we would like to thank the foundations and donors for their generous
support of the SciTech Program.
CONCLUSIONSDuring phase 1 (Figure 1), we digested wzc in pET9d, bceF in pET9d, and pET11d using restriction enzymes. As shown in Figure 4, the
digestion was not completely successful for wzc and pET9d as well as pET11d. Although bceF was successfully digested, it was not
successfully ligated unlike wzc, as seen in Figure 5 and Table 1. In phase 2 (Figure 2) the optimal expression conditions were found to be
inducible expression (pET11d) at 37°C as seen in Figure 6. In the purification phase (Figure 3) we used a His-Trap affinity column (Figure 7)
and a Size Exclusion Column (Figure 8) and achieved a large amount of protein, yet not sufficiently pure (there is no single band of Wzc,
bands of smaller proteins appear as well). From Figure 8 that Wzc has a tendency to aggregate which affects its ability to crystallize.
MATERIALS AND METHODS
RESULTS
Affinity columnHis-Trap column
Cell membrane lysis
French press
Wzc expressed from pET11d vector induced with 1mM IPTG at 37°C
PHASE #3 – PURIFICATION OF Wzc
Size exclusion column
Figure 1: Flowchart of methods of Phase #1 –
Subcloning of bceF and wzc into pET11d
Cell membrane lysis
Sonicator
Protein expressionpET9d 37°C
pET11d 37°C 1mM IPTG
pET11d 16°C 0.1mM IPTG
Transformation to BL-21
wzc in pET9d wzc in pET11d
PHASE #2 – OPTIMIZATION OF
THE EXPRESSION
Protein gel
Figure 2: Flowchart of methods of Phase #2 –
Optimization of the Expression
Figure 3: Flowchart of methods of Phase #3 –
Purification of Wzc
Figure 4: Gel results of the vector exchange of bceF
and wzc from pET9d into pET11d. All samples were cut
with BamHI-HF and NcoI-HF and ran in 1% agarose gel.
Figure 5: Colony PCR gel results of the ligation of wzc
into pET11d. C1 refers to colony 1 etc. CTL 1, 2, and 3
stand for, respectively, pET11d in XL1, wzc in pET9d,
and no bacteria cells. All samples ran in 1% agarose gel.
SAMPLEN° OF
COLONIES
WZC in pET11d in XL-1(CIP) (regular concentration)
10
WZC in pET11d in XL-1(CIP) (concentrated)
46
WZC in pET11d in XL-1(CIP) (diluted)
1
Control 1 - WZC + pET11d in XL-1 (without ligase) (CIP)
62
Control 2 - WZC + pET11d in XL-1(without ligase and insert) (CIP)
46
XL-1 colonies
Table 1: Number of colonies in
different concentrations of wzc an bceF
in pET11d transformed in XL-1.
TransformationE. Coli strain XL-1
Ligation into pET11d
T4 DNA Ligase
Digestion via restriction enzymes
CIP
PHASE #1 – SUBCLONING OF wzc
AND bceF INTO pET11d
Figure 7: Affinity column gel results of Wzc
with coomassie stain on SDS-PAGE 12% acrylamide
Figure 8: Size column gel results of Wzc
with coomassie stain on SDS-PAGE 12% acrylamidePHASE #4 – CRYSTALLIZATION
Figure 6: Gel results of Wzc in pET9d and pET11d with coomassie stain
(A) on SDS-PAGE 12% acrylamide and Western-blot results (B) of same
gel with antibody anti-His-HRP (Thermo) 1:1000
10
0b
p m
arke
r
1 K
b m
arke
r
10,0008,0006,0005,0004,000
3,000
2,5002,000
1,500
1,000
750
3,000
1,500
1,000900800700600
500
400
300
500
250
200
wzc
+ p
ET9
d
pET
11
d
bce
F +p
ET9
d
pET
11
d
10
0b
p m
arke
r
1 K
b m
arke
r
10,0008,0006,0005,0004,0003,0002,5002,000
1,500
1,000
750
3,000
1,500
1,000900800700600500
400
300
500
250
200
C1
C2
C3
C4 C5
C1
C2
C3
CTL
1
CTL
2
CTL
3
Wzc in pET11d in XL1 (CIP) concentrated
Wzc in pET11d in XL1 (CIP) regular
wzc
pET11d
mw
(kD
a)
~25
~35
~48
~63
~75 kDa
Wzc
pET
9d
SU
P 3
7C
pET
9d
pel
let
37
C
pET
11
d S
UP
1
6C
0h
r
pET
11
d p
elle
t 1
6C
0h
r
pET
11
d S
UP
1
6C
20
hr
pET
11
d p
elle
t 1
6C
20
hr
pET
11
d S
UP
3
7C
0h
r
pET
11
d p
elle
t 3
7C
0h
r
pET
11
d S
UP
3
7C
1h
r
pET
11
d p
elle
t 3
7C
1h
r
pET
11
d S
UP
3
7C
2h
r
pET
11
d p
elle
t 3
7C
2h
r
pET
11
d S
UP
3
7C
4h
r
pET
11
d p
elle
t 3
7C
4h
r
A
mw
(kD
a)
~25
~35
~48
~63
~75 kDa
Wzc
pET
9d
SU
P 3
7C
pET
9d
pe
lle
t 3
7C
pET
11
d S
UP
1
6C
0h
r
pET
11
d p
elle
t 1
6C
0h
r
pET
11
d S
UP
1
6C
20
hr
pET
11
d p
elle
t 1
6C
20
hr
pET
11
d S
UP
3
7C
0h
r
pET
11
d p
elle
t 3
7C
0h
r
pET
11
d S
UP
3
7C
1h
r
pET
11
d p
elle
t 3
7C
1h
r
pET
11
d S
UP
3
7C
2h
r
pET
11
d p
elle
t 3
7C
2h
r
pET
11
d S
UP
3
7C
4h
r
pET
11
d p
elle
t 3
7C
4h
rB
A4 A5 A6 B1 B2 B3 B4 B5 B6 C1
Fractions
~25
~35
~48
~63
~75 kDa
mw
(kD
a)
~25
~35
~48
~63
C1A5 A6 A7 A11 B3 B8 B9 B10 B12
Fractions
A12 B1 B2 B11
~75 kDa
Wzc
Wzc
