Appendix: Methods for
Isolation and Crystallization of PvdJp2, a Non-Ribosomal Peptide Synthetase Domain in
Pseudomonas aeruginosa
Betsy Ramirez and Audrey Lamb
For full article, see ugresearch.ku.edu/student/share/jur
Methods
Preparation of Plasmid
In order to produce a plasmid that contains PvdJp2-protein with an N-terminal His tag,
the PvdJ gene was amplified from P. aeruginosa pAO1 genomic DNA by PCR by use of
Herculase II supplemented with 0-10% DMSO. The forward primer
(5’ATCAGTCATATGCAGCAGGTCTACGTGGCGC 3’) contains an NdEI site (underlined).
The reverse primer (5’ATTAGAAAGCTTTTAGGAAATCAGTTTTTCAAGTTCATCG 3’)
contains a HindIII site (underlined). Digestion of the resulting amplified 336 base pair fragment
was accomplished with NdeI and HindIII and ligated into pET28 plasmid with the same
enzymes. The resulting plasmid encodes the PvdJp2 gene with an N-term His tag (N-ℎ𝑖𝑠6 PvdJp2). The plasmid was prepared by Dr. Kathleen Meneely.
N-PvdJp2 Transformation Procedure
Transformation procedures were conducted using the basic transformation procedure
(Invitrogen). One Shot® cells were thawed on ice and mixed with 1µL N-ℎ𝑖𝑠6 -PvdJp2 plasmid
DNA by gently tapping. Cells were incubated on ice for 30 minutes. The vial was placed in a
42°C heat block for exactly 30 seconds in order to heat shock the cells, and removed directly
onto ice. 250µL pre-warmed SOC medium was added aseptically to the vial, and the resulting
solution was shaken (225rpm) for 1 hour at 37°C. 50µL of the transformation reaction was plated
onto a pre-warmed LB Kanamycin plate and incubated overnight at 37°C and then stored at 4°C.
In order to prepare a glycerol stock, one transformed colony was grown in 5mL LB broth
containing 50µg/mL Kanamycin overnight at 37°C while being shaken (225rpm). 500µL of
broth was removed and combined in a 1:1 ratio with glycerol, vortexed, and stored at -80°C.
N-PvdJp2 Overexpression Trials
Glycerol stock was used to prepare a 5mL overnight culture of E. coli BL21 (DE3) cells
containing the N-ℎ𝑖𝑠6 -PvdJp2 expression plasmid. The cells were grown in LB broth containing
50µgram/mL Kanamycin at 37ºC while being shaken (225 rpm). 500µL of the cell suspension
was then added to 14 flasks of 50mL broths. Seven flasks contained 50mL of LB broth with
50µgram/mL Kanamycin, and seven additional flasks contained 50mL of Terrific Broth (TB)
with 50µgram/mL Kanamycin. The cells were shaken at a temperature of 37°C and 225rpm until
they reached log phase. When an Optical Density (O.D.) of approximately 0.7 was reached,
Table 1. Conditions tested during Induction Test
Trials:
1
2
X
X
3
4
5
6
7
8
9
X
X
10
11
X
X
TemperaturePost-induction
15°C
30°C
X
X
12
13
14
37°C
X
X
X
X
X
X
Induction
(IPTG)
0µM
X
200µM
X
X
X
X
X
X
800µM
X
X
X
X
X
X
X
Broth
LB
X
X
X
X
X
TB
X
X
X
X
X
X
X
X
X
28 Conditions were tested for N-PvdJp2. Each condition listed above was checked after 4
hours incubation and after 22 hours incubation.
Kanamycin, and 7 additional flasks contained 50mL of Terrific Broth (TB) with 50µgram/mL
the cells were subjected to varying temperatures and the addition or absence of isopropyl-BetaD-Thiogalactopyranoside (IPTG). Table 1 depicts the conditions tested, which were tested at
both 4 hours and 22 hours of incubation. After the designated incubation time had elapsed, 1mL
portions of the cells were centrifuged for 10 minutes at 5000rpm at 4ºC. The supernatant was
aspirated and the pellet collected and frozen.
In order to disrupt the cell membranes, the cells were subjected to Bug Buster protocol
(Novagen) once thawed. The thawed pellet was resuspended in 300µL of 1:100 Bug Buster
Protein Extraction Reagent and 25mM Tris pH 8 solution. 1µL of DNase was added to the cells
grown in LB broth, and 2µL of DNase was added to the cells grown in Terrific Broth. The cells
were incubated on ice for 45 minutes, and then at room temperature for the following 20 minutes
while being shaken periodically. The cells were centrifuged at 12000rpm at 4ºC for 20 minutes,
and the soluble portion collected for SDS-PAGE analysis.
N-PvdJp2 Overexpression and Purification
E. coli BL21 (DE3) cells containing the N-ℎ𝑖𝑠6 -PvdJp2 expression plasmid were grown
in LB broth containing 50µgram/mL Kanamycin at 37ºC while being shaken (225 rpm). When
an Optical Density of approximately 0.8 was reached, the temperature was reduced to 30ºC.
Protein expression was induced with the addition of isopropyl-Beta-D-Thiogalactopyranoside
(IPTG) to a final concentration of 200µM. Cells were harvested by centrifugation (5000rpm for
10 minutes at 4°C) after 22 hours. The resulting cell pellet was resuspended in 5mL of 25mM
Tris HCl pH 8, 500mM NaCl, 5mM Imidazole, and 10% glycerol (buffer A) per 4L of culture.
Cells were disrupted by use of a French Pressure cell (35000 psi) and cellular debris was
removed by centrifugation (1200g for 30 minutes at 4°C). The supernatant was applied to a
chelating sepharose fast flow column charged with Nickel Chloride and pre-equilibrated in
buffer A. N-ℎ𝑖𝑠6 -PvdJp2 eluted at in a linear gradient of 5 to 500mM imidazole in buffer A. The
pooled fractions were combined with 50mM Tris pH 8 and 150mM Sodium Citrate (Buffer B)
and then concentrated using a stir cell with an YM-3 membrane to a volume of ~ 15mL and
applied to a superdex75 size-exclusion column equilibrated with Buffer B. The fraction
containing PvdJp2-his6-N were pooled and concentrated again using the aforementioned stir cell
to 25mg/mL as determined by Bradford assay and stored at -80ºC.
Buffer Optimization Screen
320µL N-PvdJp2 at 16.6mg/mL was combined with 360µL 50% PEG 8K and incubated
for 15 minutes at room temperature. 2µL of varying salt/buffer conditions were applied to 18µL
aliquots of PEG/protein solution. This mixture was incubated for 20 minutes at room
temperature, and then centrifuged at 12000rpm for 4 minutes at room temperature. 5µL of
protein solution was added to 995µL of Bradford reagent, and the absorbance at 595nm was
measured. Conditions tested and subsequent results pictured below (table 3).
Hanging Drop Crystallization
Crystallization trials were run using the hanging drop method. Protein was suspended in a
1:1 ratio with well solution on a siliconized cover-slip and hung as a droplet over 750µL well
Protein
Buffer
PvdJp2
50mM Tris pH
Concentration
8mg/mL
Temperature (°C)
Wizard I
Wizard II
Wizard III
Wizard IV
PegRx I
PegRX 2
Peg/Ion
Salt RX 1
Salt RX 2
Index
Crystal Screen
Crystal Screen 2
MCSG-1T
MCSG-2T
solution. The crystal screens tested are diagrammed in Table 2.
25
X
8, 150mM
Sodium Citrate,
10% glycerol
50mM Tris pH
10mg/mL
X
X X
X
8, 150mM
Sodium Citrate,
10% glycerol
50mM Tris pH
25mg/mL
8, 150mM
Sodium Citrate,
Table 2. Crystal
trials for N-PvdJp2
10% glycerol
X
XX X X
X
X X X