S1
Supporting Information
Enzyme-Polyelectrolyte Complexes Boost the Catalytic Performance of
Enzymes
Martin J. Thiele1§, Mehdi D. Davari1§, Melanie König1, Isabell Hofmann1, Niklas
O. Junker2, Tayebeh Mirzaei Garakani1, Ljubica Vojcic1,3, Jörg Fitter2,4, Ulrich
Schwaneberg1,5*
1Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany
2I. Physikalisches Institut (IA), AG Biophysik, RWTH Aachen, Sommerfeldstrasse 14,
52074 Aachen, Germany
3Codexis, Inc., 200 Penobscot Drive, Redwood City, CA 94063 United States
4Institute of Complex Systems (ICS-5): Molecular Biophysics, Forschungszentrum
Jülich GmbH, 52425 Jülich, Germany
5DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056
Aachen, Germany
*Corresponding author: [email protected]
,Tel.: +49 241 80 24170, Fax: +49 241 80 22387
S2
Content Description Page Detailed explanation of Material and Method
Table S1 Method M1 Figure S1 Method M2
The composition of 15°dGH water solution protease 1ST3 purification via ion exchange chromatography (IEC) Solubility assay of the purified protease 1ST3 Labelling of protease 1ST3 and size exclusion chromatography (SEC)
S4 S5-S6
S7 S8
Method M3 Method M4 Method M5 Method M6
Fluorescence correlation spectroscopy (FCS) Isothermal Titration Calorimetry (ITC) Site-saturation Mutagenesis Library Generation Site-directed Mutagenesis protease 1ST3 Variant Generation
S9 S10 S11 S12
Method M7 CD spectroscopy (CD) S13 Method M8 Models and force field parameters for PAA and γ-PGA S14
Supporting Data and Figures
Figure S2
Effect of pH on protease 1ST3 activity in the presence and absence of polyelectrolytes PAA and γ-PGA
S15-S16
Figure S3
Performance test of the crude supernatant with and without expressed active protease
S17
Figure S4 The effect of concentration of the polyelectrolytes on the protease 1ST3 activity
S18
Figure S5
Proteolytic performance of the purified protease 1ST3
in the presence of PAA and γ-PGA in 15° and 0°dGH
water
S19
Figure S6
Proteolytic activity of the purified unlabeled and labeled protease
S20
Figure S7
Determination of protease 1ST3 radius of gyration (Rg) by using molecular dynamics simulations
S21
Figure S8
The effect of length of PAA and γ-PGA on the
proteolytic performance of the protease 1ST3
S22-S23
Figure S9 Table S2
Determination of the relative micro-viscosity of γ-PGA
Calculated hydrodynamic radii of the protease-γ-PGA
complex
S24 S25
Figure S10 ITC measurement to determine binding thermodynamics of protease-PAA interaction
S26
Figure S11 ITC measurement to determine the background titration heat
S27
Table S3 Thermodynamic parameters obtained from ITC S28 Figure S12
Mean square fluctuation (RMSF) of protease 1ST3 residues interacting with PAA and γ-PGA
S29
Figure S13
Far-UV CD spectra of the protease 1ST3 in the presence of PAA and γ-PGA
S30
Figure S14 Protease 1ST3 and PAA: MD simulation (1) S31 Figure S15 Protease 1ST3 and PAA: MD simulation (2) S32 Figure S16 Protease 1ST3 and γ-PGA: MD simulation (1) S33
Figure S17 Protease 1ST3 and γ-PGA: MD simulation (2) S34
Figure S18 Effect of ionic strength on protease 1ST3 bossting in
the presence of polyelectrolytes PAA and γ-PGA.
S35
Figure S19 Electrostatic potential distribution on protease surface upon binding of PAA
S36
Figure S20 Number of contacts between PAA chains (chain 1 and chain 2) and protease 1ST3 along MD simulation trajectory
S37
Table S4 Primers used for site-saturation mutagenesis (SSM) of S38
S3
the protease 1ST3 gene Table S5 Primers used for site-directed mutagenesis (SDM) of
the protease 1ST3 gene S39
S4
Detailed explanation of Material and Method
Table S1: The composition of 15°dGH water solutions; dGH stands for degrees of
general hardness.
15°dGH* (Ionic strength in 10.5 mmol L-1)
Inorganic salt g L-1 mol L-1
CaCl2·2H2O 0.29 0.001974
MgCl2·6H2O 0.14 0.000689
NaHCO3 0.21 0.002500
*ultrapure water from arium ®pro UV|DI water purification system
S5
M1: Protease 1ST3 purification via ion exchange chromatography (IEC)
The expression of the protease 1ST3 in the high expression vector pHKL was
accomplished in shaking flasks (500 mL) using 100 ml LB media (900 rpm, 37 °C, 48
h, and 70 % humidity). A clear supernatant was obtained after centrifugation
(Eppendorf 5810R, 14000×g, 30 min, 4 °C) of the cell culture supernatant in a 50 ml
eppendorf conical tube. Afterwards, the supernatant was loaded into an Amicon
Ultra-15 centrifugal filter unit (10 kDa MWCO; Merck Millipore) to concentrate the
supernatant to 5 mL final volume and to exchange the LB media with HEPES buffer
(pH 7.0, 20 mM). The concentrated supernatant was loaded into a cation exchange
chromatography column (GE Healthcare HiTrap SP HP cation exchange
chromatography column (5 ml) equilibrated with HEPES buffer (pH 7.0, 20 mM)). For
elution of the protease, the cation exchange column was disconnected and a linear
gradient of sodium chloride up to 1 M in HEPES buffer (pH 7.0, 20 mM) was used to
elute the absorbed proteins from the cation exchange column. The peak fractions
were analyzed by SDS-Page and pooled together (Figure S1 A). Protein
concentration of the purified protease 1ST3 was normalized by measuring the total
protein concentration using the Bradford protein assay. The pooled purified protein
fractions were analyzed with ExperionTM Automated Electrophoresis System and
revealed that the protease 1ST3 was obtained with ≥ 85 % purity (Figure S1 B). In
order to ensure proteolytic performance of the purified protease 1ST3 on the CO-3
cotton surface, a solubility assay was performed (Figure S1 C).
Figure S1: A: SDS-PAGE of TCA-precipitated supernatant containing protease 1ST3 (~27
kDa) and protease 1ST3 purified via ion-exchange chromatography (~27 kDa). B: The
10 20 30 40 50 600.00
0.02
0.04
0.06
0.08
0.10
0.12
Ab
so
rban
ce
Time [min]
Protease purified
Protease supernatant
Protease
M Supernatant Purified
CA
~70-~55-~40-
~35-
~25-
B
M Protease
S6
pooled purified protein fractions were analyzed with ExperionTM Automated Electrophoresis
System (BIO-RAD, Munich, Germany) and revealed that the protease 1ST3 was obtained
with ≥85 % purity. C: Solubility performance of purified protease 1ST3 (9x10-6 mol L-1 grey
bars) and supernatant containing protease 1ST3 (blue bars) over the time in 15°dGH water
at 40°C.
S7
M2: Labelling of protease 1ST3 and size exclusion chromatography (SEC)
The protease 1ST3 was labeled with DyLight 650 amine-reactive dye according the
protocol supplied by the vendor (Thermo Scientific). DyLight 650 is activated with an
N-hydroxysuccinimide (NHS) ester moiety to react with surface exposed amino acid
groups of lysine residues to form stable amide bonds. The reaction was performed in
PBS buffer (phosphate-buffered saline, pH 8.5) and the excess of unreacted dye and
protease 1ST3 were separated through size exclusion by using a Sephadex G-25 gel
medium (GE Healthcare) for gel filtration chromatography. After filtration, the degree
of labeling (DOL, dye-to-protein ratio) was determined by absorption spectroscopy
and calculated according to the vendors protocol (Thermo Scientific). For the
subsequent FCS measurements, the labelled protease 1ST3 was highly diluted in
15°dGH water.
S8
M3: Fluorescence correlation spectroscopy (FCS)
FCS measurements were performed with a PicoQuant (Berlin, Germany) MicroTime
200 confocal microscope equipped with a red (640 nm) diode laser and an
UPLSAPO 60x/1.2NA objective from Olympus (Shinjuku, Japan). The emitted
photons were collected by the microscope objective and passed through a dual-band
dichroic mirror (Omega 475-625DBDR, Brattleboro, USA). Subsequently, they were
focused on a pinhole and splitted into two detection channels by a 50/50 beam
splitter cube from Linos Photonics (Göttingen, Germany). The obtained photons were
filtered by a bandpass emission filter (Chroma Technology HQ690/70M) and focused
on two Perkin Elmer (Waltham, USA) SPAD detectors (SPCM-CD3077-H and
SPCM-AQR-14). The lasers were operated at a frequency of 20 MHz by means of a
computer controlled PicoQuant PDL828 ”Sepia-II” laser driver. Photon counts were
processed with a”PicoHarp-300” time correlated single photon counting (TCSPC)
acquisition unit from PicoQuant. In the case of FCS, we employed a sample
concentration in the nanomolar regime. In order to reduce effects from the refractive
index mismatch, typically occurring in high concentrated solutions, we used a small
30µm pinhole and a limited coverslip-focus-distance of 10µm. FCS curves were
generated and analyzed with the help of the software Symphotime64 from
PicoQuant. The calculated autocorrelation function was fitted by a model
(eq.S1)
considering two translational diffusion components and triple state dynamics. Here,
<N> denotes the average number of particles in the effective volume, 𝜌𝑖 the fraction
of molecules diffusing with the respective translational diffusion time 𝜏𝐷,𝑖 ,Tr the
fraction of molecules in the triplet state and 𝜏𝑇 the triplet relaxation time. The
parameter 𝜅 describes the ratio of the axial to the radial dimension of the effective
volume and needs to be determined in a calibration measurement performed under
the same conditions as the actual experiment.
2
1/21
2
, ,
11( )
11 1
T
ir r
ir
D i D i
T T eG
N T
S9
The effective volume was calibrated with the help of a sample with a known diffusion
coefficient D
(eq. S2)
Here, 𝜔0 denotes the lateral 𝑒−2 radius of the effective volume. The hydrodynamic
radius 𝑅𝐻 of the diffusing fluorescent molecule is related to the translational diffusion
coefficient by the Stokes-Einstein equation:
(eq. S3)
𝑘𝐵 denotes Boltzmann’s constant, T the temperature in Kelvin, 𝜂 the microscopic
viscosity of the solvent. The microscopic viscosities of different polymer solutions
were estimated by FCS studies with Alexa 647 fluorophores, freely diffusing in the
respective polymer solutions (see Figure S6).
0
4D
D
6
BH
k TR
D
S10
M4: Isothermal Titration Calorimetry (ITC)
ITC was used to quantify the driving force of polyelectrolyte-protease 1ST3
interaction by measuring the enthalpy change when both species interact, which
enables the determination of thermodynamic parameters. ITC was carried out on
TAM III (TA Instruments, Germany) after gentle degassing of all samples for 10 min
at room temperature and 635 mm Hg to remove CO2 from the working solutions. For
the measurement, concentrated PAA solution (5.5x10-4 mol L-1 in 15°dGH water) was
titrated into 6x10-5 mol L-1 purified protease 1ST3 solution (diluted in 15°dGH water)
to determine binding isotherms at low polymer concentration. Titrations
measurements were performed at 25°C in triplicates and the system was first allowed
to equilibrate for at least 6 h. In total, 200 µl of 5.5x10-4 mol L-1 aqueous PAA solution
was filled into a glass syringe followed by removing gas bubbles inside the syringe.
Then, 500 μL of 6x10-5 mol L-1 protease 1ST3 solution was filled in the ampoule of
stainless steel and the PAA solution titrated (40 injections of 5 μl) into the protease
1ST3 solution. As reference, 200 µl pure 15°dGH water was titrated into the protease
1ST3 solution. Injections were performed for 10 s with 20 min between injections
allowing the signal to return to the baseline. Titration data were analyzed (i.e.
automated baseline adjustment and peak integration) and fitted by using Origin 7.0
software 1. Values of stoichiometry (n), association constant (Ka) and enthalpy (ΔH)
were obtained from the fit, whereas Gibbs free energy (ΔG) and entropy (-TΔS) were
obtained by the following equations2.
ΔG=−RTln(Ka) (eq. S4a)
ΔG=ΔH-TΔS (eq. S4b)
Where R is the gas constant (8.3145 J K−1·mol−1) and T is the absolute temperature
in K.
S11
M5: Site-saturation Mutagenesis: Library Generation
Site-saturation mutagenesis of protease 1ST3 was performed at 9 selected positions
on protease 1ST3 in the commercial shuttle vector pHY300PLK (Takara Bio Inc,
Japan).3 For the site-saturation mutagenesis PCR, a thermal cycler (Eppendorf
Mastercyler proS, Hamburg, Germany) (First stage: 98°C for 30 s, 1 cycle; 98°C 10
s/gradient 55-65°C, 30 s/72°C, 4 min, 4 cycles. Second stage: 98°C for 30 s, 1 cycle;
98°C, 10 s/gradient 55-65°C, 30 s/72°C 4 min, 24 cycles; 72°C for 10 min, 1 cycle),
PhuS DNA Polymerase (2 U), 0.20 mM dNTP mix, and 0.2 μM of each NNK primer
(primer sequences are shown in Table S4) together with template (~20 ng;
pHY300protease_WT) were used. After the PCR, DpnI (20 U; New England Biolabs)
was supplemented for digestion of maternal template DNA and incubated overnight
at 37 °C. The DpnI-digested PCR products were purified by using a NucleoSpin®
Extract II Purification Kit (Macherey-Nagel, Düren, Germany), transformed into
Escherichia coli DH5α (E. coli DH5α) and plated on LB agar plates containing 100
mg mL-1 ampicillin.4 The E. coli DH5α mutants were cultivated (37°C, 24 h) and
afterwards plasmids from approximately 200 single pooled E. coli colonies isolated
using a NucleoSpin Plasmid Kit (Macherey-Nagel) followed by transformation into
protease-deficient Bacillus subtilis DB104 strain (nprR2 nprE18 and ΔaprA3) 5 using
a natural competence based transformation method and plated on LB agar plates
supplemented with 2% (w/v) skim milk and 15 µg mL-1 tetracycline.6 Skim-milk
indicator agar plates were used to select protease 1ST3 variants showing proteolytic
activity.7 The generated libraries were screened in 15°dGH water with and without
PAA (5.5x10-4 mol L-1) or γ-PGA (1.7x10-5) at 40°C and improved protease 1ST3
variants compared to the WT protease 1ST3 analyzed and sequence analyzed.
S12
M6: Site-directed Mutagenesis: protease 1ST3 Variant Generation
Site-directed mutagenesis was performed at 15 selected positions on the protease
1ST3 in the industrial expression vector pHKL (provided by Henkel AG & Co. KGaA,
Düsseldorf, Germany) suitable for high level extracellular protease 1ST3 production
in B. subtilis DB104.8-9 The PCR template pHKLprotease_WT was first methylated
with Dam methyltransferase (New England Biolabs) according to the manual, to
guarantee complete DpnI (New England Biolabs) digestion of the used
pHKLprotease_WT template DNA. For the site-directed mutagenesis PCR, a thermal
cycler (Eppendorf Mastercyler proS, Hamburg,Germany) (First stage: 98°C for 30 s,
1 cycle; 98°C 10 s/gradient 55-65°C, 30 s/72°C, 4 min, 4 cycles. Second stage: 98°C
for 30 s, 1 cycle; 98°C, 10 s/gradient 55-65°C, 30 s/72°C 4 min, 24 cycles; 72°C for
10 min, 1 cycle), PhuS DNA Polymerase (2 U), 0.20 mM dNTP mix, and 0.2 μM of
each SDM primer (primer sequences are shown in Table S5) together with template
(~20 ng; dam methylated pHKLprotease_WT) were used. After the PCR, DpnI (20 U;
New England Biolabs) was supplemented for digestion of maternal template DNA
and incubated overnight at 37 °C. The DpnI-digested PCR products were purified by
using a NucleoSpin® Extract II Purification Kit (Macherey-Nagel, Düren, Germany)
and transformed via protoplastation into a protease-deficient and poly(L-γ-glutamic
acid)-deficient B. subtilis DB104.1 strain (provided by Henkel AG & Co. KGaA,
Düsseldorf, Germany) and plated on LB agar plates containing 50 mg mL-1
kanamycin.10
S13
M7: CD spectroscopy (CD)
CD spectra were recorded on CD-Photometer JASCO J-1100 (JASCO Germany
GmbH) at room temperature using 0.2 mm path length cuvettes and 1.5 mg/ml
protease 1ST3 concentration. The bandwidth of 1 nm, data pitch of 0.2 nm, scan
speed of 100 nm/min and data integration time (DIT) of 0.5 s was used for all
measurements. An average of three spectra was taken for all measurements. The
resulting CD spectra were analyzed by Spectra Manager II Spectroscopy Software
Suite. For the CD measurements, the sole protease 1ST3 and protease 1ST3 in the
presence of PAA or γ-PGA and was pre-incubated for 5 min in 15°dGH water and
subsequently measured in 3 replicates.
S14
M8: Models and force field parameters for PAA and γ-PGA
For PAA and γ-PGA, polybuild software11 was used to build structural models of fully-
deprotonated single polymer chains in atactic configuration with 20 repeating units
(PAA (pKa: 5.9812) or γ-PGA (pKa: 4.8613)). The structural models of PAA and γ-PGA
monomers are shown Figure 1. PAA and γ-PGA models were parameterized by using
Gaussian09 software package and R.E.D. server, respectively.14-15 For
parameterization, the 20-mer structural model of PAA and γ-PGA was split up into
head group, tail group and repeating units. In case of the PAA model, head and tail
group were capped with one methyl group, the repeating units with two methyl
groups. For the γ-PGA model, the negatively charged repeating unit was capped with
a zwitteriinoic head group and and double negatively charged tail group. RESP
charges were derived using the R.E.D. server with charge constraints on the capping
groups. Similarly, the zwitterionic head and the twice negatively charged tail group
were parametrized: Both were capped with one ACE and one NME group each.
However, for calculating charges a second molecule is necessary, i.e. in case of the
head group methylammonium (CH3NH3+) is used together with intermolecular charge
constraints between methylammonium and the ACE-NH group of the head group.
Additionally, an intramolecular charged constraint has to be applied to the NME cap.
For the tail group, acetate (CH3COO–) is used together with intermolecular charge
constraints between acetate and the CO-NME group of the tail group. Here, the
intramolecular charge constraint has to be applied to the ACE cap. Each group
carries one negative charge resulting in a net charge of –20. Geometry optimizations
were performed by applying density functional theory (DFT) with B3LYP functional16-
18 and 6-31G (d,p) basis set19-20. In order to assign partial charges, electrostatic
potential (ESP) charges were calculated using the Hartree-Fock (HF) method and 6-
31G (d) basis set19-20.19-20 Amber ff99SB compatible RESP charges were derived
using the Antechamber module21 of AmberTools1422. Furthermore, the ACPYPE23
software was used to get a GROMACS compatible topology for PAA or γ-PGA with
AMBER99SB atom types.
S15
Supporting Data and Figures
Effect of pH on protease 1ST3 activity in the presence and absence of
polyelectrolytes PAA and γ-PGA
Experiment was performed in 15°dGH water with varied pH from 4-11 in which the
effect of pH on protease activity in the presence and absence of polyelectrolytes PAA
(pKa: 5.9812) or γ-PGA (pKa: 4.8613) was quantified in two sets of experiments (one
with polyelectrolyte and one without). The values are given in proteolytic activity
values in slope min-1 to compare the polyelectrolyte boosting effect (Figure S2).
Figure S2: Effect of pH on protease 1ST3 activity in the presence and absence of
polyelectrolytes PAA and γ-PGA. Proteolytic activity (slope min-1) of the protease 1ST3
supernatant (1:5 dilution, black circle) in the presence of PAA (blue circle) or γ-PGA (red
circle) using the Suc-AAPF-pNA assay across 10 min incubation. Sole PAA (black triangle)
and γ-PGA (black square) polyelectrolytes revealed no activity toward Suc-AAPF-pNA
substrate.
As shown in Figure S2, the protease 1ST3 activity was measurable over a broad pH
range while the optimal pH lies more in the alkaline range. In the presence of PAA
and γ-PGA, the boosting on the protease 1ST3 was only observed above pH 7.5
(Figure S2). At low pH (<7), proteolytic activity is sharply reduced in the presence of
both polyelectrolytes indicating that the conformation, protonation/deprotonation state
and association with divalent ions (e.g. Ca2+) of both polyelectrolytes in aqueous
solutions effects the proteolytic activity of the protease 1ST3. Nevertheless, at pH >7,
4 5 6 7 8 9 10 11
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Pro
teo
lyti
c a
cti
vit
y
tow
ard
Su
c-A
AP
F-p
NA
[A
U4
10
nm
min
-1]
pH
PAA
-PGA
Protease
Protease + PAA
Protease + -PGA
S16
γ-PGA (pKA: 4.86) and PAA (pKa: 5.98) are fully deprotonated and exhibit very likely
well exposed negative charges which are favorable to interact with the protease
1ST3 surface causing an activity boost.”
S17
Performance test of the crude supernatant with and without expressed active
protease 1ST3
In consideration of possible unspecific site effects of several other secreted proteins
present in the crude supernatant, an engineered proteolytically inactive protease
1ST3 variant (S215Y) was used as a control. Performance tests of the supernatant
containing S215Y protease 1ST3 variant by using solubility assay at 40°C was
performed and showed no significant performance compared to the supernatant with
the wild type (WT) protease 1ST3 over the time on the CO-3 cotton surface
confirming no significant proteolytic performance of the crude supernatant (Figure
S3).
Figure S3: Solubility performance of supernatant (1:5 dilution) containing WT protease 1ST3
(dark grey bars) and protease 1ST3 variant S215Y (light dashed grey bars) after 10 and 60
min incubation in 15°dGH water at 40°C.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
60
Time [min]
Protease S215Y
Protease WT
Pro
teo
lyti
c p
erf
orm
an
ce
[AU
50
0n
m]
10
S18
The effect of concentration of the polyelectrolytes on the protease 1ST3
activity
To identify the highest boosting of protease in the presence of polyelectrolytes, we
have measured the protease activity in different concentrations of PAA and γ-PGA
polyelectrolytes. In Figure S4 A-B, the proteolytic activity of the protease 1ST3 in the
presence of different concentrations of PAA and γ-PGA polyelectrolyte is depicted. As
can be seen in Figure S4 A-B, the highest observable boost on the proteolytic
activity was achieved in the presence of 1.1x10-4 mol L-1 PAA or 3.3x10-6 mol L-1 γ-
PGA. Therefore, polyelectrolyte concentration 1.1x10-4 mol L-1 for PAA or 3.3x10-6 mol
L-1 for γ-PGA were chosen for further measurements and characterization of the
protease boosting effect on complex substrates (skim milk and CO-3), respectively.
Figure S4: Proteolytic activity in the presence of different concentrations of polyelectrolytes
PAA and γ-PGA. A-B: Proteolytic activity (slope min-1) of sole PAA (blue filled triangle) and γ-
PGA (red filled triangle) polyelectrolytes, protease 1ST3 supernatant (1:5 dilution, filled grey
circle) in the presence of PAA (open grey circle) or γ-PGA (open grey circle) using the Suc-
AAPF-pNA assay across 10 min incubation.
0.0 2.0x10-4
4.0x10-4
6.0x10-4
8.0x10-4
1.0x10-3
1.2x10-3
0.00
0.01
0.03
0.04
0.05
0.06
0.07
Pro
teo
lyti
c a
cti
vit
y
tow
ard
Su
c-A
AP
F-p
NA
[Slo
pe m
in-1]
PAA concentration [mol L-1]
Protease
PAA
Protease + PAA
0 1x10-5
2x10-5
3x10-5
4x10-5
0.00
0.01
0.03
0.04
0.05
0.06
0.07
Pro
teo
lyti
c a
cti
vit
y
tow
ard
Su
c-A
AP
F-p
NA
[Slo
pe m
in-1]
-PGA concentration [mol L-1]
Protease
-PGA
Protease + -PGA
A B
S19
Proteolytic performance of the purified protease 1ST3 in the presence of PAA
and γ-PGA
In order to examine specificity of the observed improved proteolytic activity in the
presence of PAA and γ-PGA polyelectrolytes, the protease 1ST3 was purified via ion
exchange chromatography and its proteolytic activity analyzed using the solubility
assay. The measurements showed that the proteolytic performance of the pure
protease 1ST3 on the CO-3 cotton surface in the presence of 1.1x10-4 mol L-1 PAA is
strongly boosted (up to ~3.5 times) compared to the sole protease 1ST3 performance
in 15°dGH water (Figure S5 A). protease 1ST3 performance in the presence of
3.3x10-6 mol L-1 γ-PGA was also boosted (up to ~1.3 times) in 15°dGH water but at a
significantly lower level compared to PAA (Figure S3 B).
Figure S5: Normalized proteolytic activity of the 9x10-6 mol L-1 purified protease 1ST3 in the
presence of 1.1x10-4 mol L-1 PAA (A) or 3.3x10-6 mol L-1 γ-PGA (B) in 15°dGH water at 40°C
after 1 h incubation. All measured absorbance values were normalized against solubility
performance of respective aqueous solutions with and without polyelectrolytes. Dashed
arrow shows the boosting of the protease 1ST3 performance.
0.0140.008
0.089
0.0140.008
0.089
0.00
0.03
0.06
0.09
0.12
0.15
Pro
teo
lyti
c p
erf
orm
an
ce
[AU
500n
m a
fter
1h]
Individual component
Purified protease
PAA
Purified protease + PAA
0.014
0.002
0.0210.014
0.002
0.021
0.00
0.03
0.06
0.09
0.12
0.15
P
rote
oly
tic p
erf
orm
an
ce
[AU
500n
m a
fter
1h]
Individual component
Purified protease
-PGA
Purified protease + -PGA
A B
S20
Proteolytic activity of the purified unlabeled and labeled protease
Proteolytic activity of the labeled purified protease 1ST3 was measured and
compared with the unlabeled purified protease 1ST3 to analyze the influence of the
labeling process on the enzyme activity by using Suc-AAPF-pNA assay. As shown in
Figure S6, proteolytic activity of the labeled purified protease 1ST3 reveals similar
performance compared to the unlabeled purified protease.
Figure S6: Proteolytic activity (slope min-1) of the purified unlabeled protease 1ST3 in
comparison to the purified labeled protease 1ST3 with Dylight 650. Proteolytic activity of
7x10-6 mol L-1 purified unlabeled protease 1ST3 (square) and 7x10-6 mol L-1 purified labeled
protease 1ST3 (circle) was measured by using the Suc-AAPF-pNA assay. Sole Suc-AAPF-
pNA substrate (triangle) serves as control and showed no cleavage over the time.
0 100 200 300 400 500
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Protease unlabeled
Protease labeled
Control
Pro
teo
lyti
c a
cti
vit
y
[AU
410n
m m
in-1]
Time [sec]
S21
Determination of protease 1ST3 radius of gyration (Rg) by using molecular
dynamics simulations
Figure S7: Change of the radius of gyration Rg of protease 1ST3 during MD simulation over
time for all 3 independent production runs.
S22
The effect of length of PAA and γ-PGA on the proteolytic performance of the
protease 1ST3
Boosting analysis on the protease for CO-3 substrate by using different lengths of
PAA revealed a clear dependency of the boosting effect on the PAA polymer length.
As shown in Figure S8 A, the boosting of the protease in the presence of 15 kDa and
100 kDa PAA can be observed but at a lower level compared to the 4.5 kDa PAA. In
contrast to PAA, boosting analysis of the protease for CO-3 substrate by using
different lengths of γ-PGA revealed no dependency on the polymer length (Figure S8
B). We assume that longer PAA and γ-PGA chain lengths undergo a conformational
change from an extended into a coiled conformation. The coiled PAA and γ-PGA
polymers might lose its interaction with protein surface.
Interestingly, it has been reported for PAA that polymer lengths <15 kDA adapt an
extended conformation and do not exhibit conformational change into a coiled
structure in contrast to polymer lengths >15 kDA 24. Additionally, structural analysis of
γ-PGA shows that at alkaline pH might adapt mostly random coiled structures 25.
Similar conformational behavior can be expected for γ-PGA with different molecular
weights. The random coiled polymer structures might have less interactions with the
protease 1ST3 and explain the low boosting ability of coiled structures of γ-PGA
(~150 kDa) and PAA (~100 kDa; Figure S8).
Figure S8: Proteolytic performance toward CO-3 substrate in the presence of varied length
of polyelectrolytes PAA and γ-PGA. Proteolytic performance of the supernatant containing
protease (1:5 dilution) after 10 min incubation in the absence and presence of A: 1.1x10-4
mol L-1 4.5 kDa PAA, 3.3x10-4 mol L-1 15 kDa PAA and 0.05x10-4 100 kDa PAA or B: 1.0x10-4
mol L-1 5 kDa γ-PGA, 0.1x10-4 mol L-1 50 γ-PGA and 0.03x10-4 150 kDa γ-PGA by using
0.000
0.006
0.012
0.018
0.024
0.030
Pro
teo
lyti
c p
erf
orm
an
ce
tow
ard
CO
-3
[aft
er
10
min
]
Individual component
Protease
Protease + 5 kDa -PGA
Protease + 50 kDa -PGA
Protease + 150 kDa -PGA
0.000
0.006
0.012
0.018
0.024
0.030
Pro
teo
lyti
c p
erf
orm
an
ce
tow
ard
CO
-3
[aft
er
10
min
]
Individual component
Protease
Protease + 4.5 kDa PAA
Protease + 15 kDa PAA
Protease + 100 kDa PAA
A B
S23
solubility assay. Dashed blue and red arrows show the boosting of the protease performance
in the presence of PAA and γ-PGA, respectively. The sole PAA and γ-PGA showed barely
solubilization efficiency on the CO-3 cotton.
S24
Determination of viscosity of γ-PGA polyelectrolyte
In order to separate effects of specific γ-PGA/protease 1ST3 binding interactions from
non-specific viscosity effects of γ-PGA on the diffusion of protease, the unknown
micro-viscosity of solutions containing different γ-PGA concentrations was
determined. For this purpose a model compound (here a freely diffusing Alexa 647
dye), which does not interact specifically with γ-PGA, was employed (Figure S9).
Figure S9: Determination of the relative micro-viscosity by measuring the free diffusing dye
Alexa 647 in solutions with different concentrations of γ-PGA. A: Diffusion coefficients for
Alexa-647 are shown for the used concentrations of γ-PGA. B: Corresponding microscopic
viscosities 𝜂 were determined from the diffusion coefficients by the relation η
η0 =
D0
D in
accordance to the Stokes-Einstein equation, based on known values of the viscosity 𝜂0 and
the diffusion 𝐷0 coefficient of Alexa 647 in pure water buffer.
10-5
10-4
10-3
150
175
200
225
250
275
300
325
D [
µm
2 s
-1]
-PGA concentration [mol L-1]
A B
10-5
10-4
10-3
1.0
1.2
1.4
1.6
1.8
2.0
-PGA concentration [mol L-1]
S25
Hydrodynamic radii of the protease-γ-PGA complex from FCS measurements
For identifying and monitoring of specific interaction between the protease 1ST3 and
γ-PGA, diffusion coefficients of the sole protease 1ST3 and the protease 1ST3 bound
to the polyelectrolyte γ-PGA were determined in solutions with increasing
polyelectrolyte concentrations. As already indicated by the autocorrelation curves
shown in Figure 3 A, fitting of the data was only possible with two diffusion
components in the case of proteases in γ-PGA solutions (see eq. S1, S2 and S3). We
obtained reasonable fits by fixing one component to diffusing proteases sensing only
viscosity effects (no specific binding to the polyelectrolyte, characterized by D2 and
RH2) and another component sensing both, viscosity effects and in addition specific
binding of γ-PGA to the protease 1ST3 (characterized by D1 and RH1). The size of the
protease 1ST3 with bound γ-PGA was determined from data measured at the highest
γ-PGA concentration (3.3x10-4 mol L-1) and the corresponding RH2-value was fixed for
the other concentrations. For details of the model fit see Method M3. The most
important fitting parameters are given in Table S2.
Table S2: Obtained fitting parameter from protease-γ-PGA complex measured in
15°dGH.
γ-PGA
Concentration [10-4 mol L-1]
0 0.033 0.066 0.165 0.33 0.66 1.33 2.0 3.3
η/η0 1 1.004 ± 0.026
1.070 ± 0.051
1.097 ± 0.023
1.142 ± 0.036
1.289 ± 0.033
1.503 ± 0.049
1.550 ± 0.041
1.949 ± 0.085
D1
[μm2/s] - 29.92 27.95 27.21 26.26 23.18 19.93 19.29
15.35 ± 1.23
RH1$
[nm] - 7.20 7.20 7.20 7.20 7.20 7.20 7.20
7.20 ± 0.45
D2
[μm2/s] 93.88 ±
0.63 94.06 87.76 85.67 82.71 73.06 62.57 60.73 48.30
RH2 *
[nm]2.29 ± 0.23
2.29 2.29 2.29 2.29 2.29 2.29 2.29 2.29
Tr 0.282 0.281 0.275 0.263 0.262 0.264 0.265 0.269 0.274
µs
14.88 14.89 14.35 12.65 12.43 13.41 13.49 13.68 13.89
*RH2 values, determined for unbound protease 1ST3 in pure buffer conditions (bold number),
were kept fixed for fits of data measured at the other concentrations.
$RH1 values, determined for bound protease 1ST3 at the highest γ-PGA concentration (bold
number), were kept fixed for data measured at the other concentrations.
S26
ITC measurement to determine binding thermodynamics of protease-PAA
interaction
In order to quantify binding between low molecular weight polyelectrolyte PAA (~4.5
kDa) and the purified protease, concentrated PAA solution (5.5x10-4 mol L-1) was
titrated into a 6x10-5 mol L-1 protease 1ST3 solution (diluted in 15°dGH water). Three
individual titrations were performed and the resulting isotherms summarized (Figure
S10).
Figure S10: Summary of ITC data from three individual titrations for the binding of 5.5x10-4
mol L-1 PAA to the 6x10-5 mol L-1 protease: A: Primary raw heat peaks of three individual
titration experiments plotted as heat flow Q (µJ sec-1) vs. time (min). B: Integrated and
concentration-normalized heat of each injection vs. the molar ratio of protease 1ST3 to PAA
in the sample cell together with the line which represents the curve fit to the binding
isothermal functions. The heat associated with the first injection is not considered for the data
analysis.
Figure S10 A depicts the binding isotherms occurred during interaction between the
protease 1ST3 and PAA. Each negative peak corresponds to heat which is released
during association of polymer molecules with the protease 1ST3 indicating an
exothermic driven assembly of protease-polyelectrolyte complexes. All injections
involve exothermic heat signals with a stepwise saturation of the protease 1ST3 via
PAA molecules (Figure S10 A). The resulting raw heat peaks from protease-
polyelectrolyte interaction were integrated as a function of the molar ratio between
the protease 1ST3 and PAA solution and fitted by using Origin 7.0 software (Figure
S10 B). Changes in Gibbs free energy (ΔG), binding enthalpy (ΔH) and entropy (-
TΔS) of the interaction between protease 1ST3 and PAA were determined (see eq.
S4 a,b) from curve-fitting analysis and summarized in Table S3.
0 200 400 600 800-8
-6
-4
-2
0
Titration I
Titration II
Titration III
Hea
t F
low
Q
[µJ s
ec
-1]
Time [min]
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
-20
-16
-12
-8
-4
0
Titration I
Titration II
Titration III
Rea
cti
on
Hea
t
[kJ m
ol-
1 o
f in
jecta
nt]
Molar ratio
A B
S27
ITC measurement to determine the background titration heat
In order to determine the background titration heat, 15°dGH water was injected into
6x10-5 mol L-1 protease 1ST3 solution. Titrations were performed with 50 injections of
5 μl water (total 250 µl) into 500 µl 6x10-5 mol L-1 protease 1ST3 solution. Injections
were carried out for 10 s with 10 min between measurements allowing the signal to
return to the baseline. As shown in Figure S11 A-B, the resulting heats of dilution
(HD) during the titration of water into the protease 1ST3 solution revealed constant
peak sizes with low isotherms (~1 µJ sec-1) in comparison to the evaluated binding
isotherms of 5.5x10-4 mol L-1 PAA titrated into 6x10-5 mol L-1 protease 1ST3 solution
(diluted in 15°dGH water).
Figure S11: A: Background titration heat (BH, red curve) of 15°dGH water into 6x10-5 mol L-1
protease 1ST3 solution (diluted in 15°dGH water) and B: Primary raw heat peaks of 15°dGH
water with 5.5x10-4 mol L-1 PAA into 15°dGH water containing 6x10-5 mol L-1 protease 1ST3
(black peaks).
0 100 200 300 400 500-8
-6
-4
-2
0
Heat
Flo
w Q
[µJ s
ec
-1]
Time [min]
Titration I
Control Titration
0 100 200 300 400 500-8
-6
-4
-2
0
Heat
Flo
w Q
[µJ s
ec
-1]
Time [min]
Control Titration
A BHD HD
S28
Thermodynamic parameters obtained from ITC
The heat of dilution of the protease 1ST3 solution during the titration with sole
15°dGH water revealed low isotherms and was negligible for further data evaluation.
The resulting isotherms were processed (see eq. S4 a,b).
and fitted to evaluate the association constant (Ka), Gibbs free energy of binding
(ΔG), binding enthalpy (ΔH) and entropy (-TΔS) considering the binding of one (with
1:1 stoichiometry) or two PAA chains on the protease 1ST3 surface (with 2:1
stoichiometry) (Table S3).
Table S3: Thermodynamic parameters from three individual titrations for the binding of PAA
to the protease 1ST3 in 15°dGH water measured at 25°C.
n (stoichiometry)
ΔG
(kJ mol-1
)
ΔH
(kJ mol-1
)
-TΔS
(kJ mol-1
)
1:1 -2.8 ± 0.2 -18.2 ± 0.7 15.5 ± 0.9
2:1 0.7 ± 0.1 -18.2 ± 0.7 18.9 ± 0.7
S29
Analysis of amino acids flexibility during the MD simulations
Comparison of root mean square fluctuation (RMSF) of protease 1ST3 residues
interacting with PAA and γ-PGA during the MD simulations is shown in Figure S12.
Figure S12: RMSF of protease 1ST3 aminoacid residue for production runs during MD
simulation in water, protease-PAA and protease-γ-PGA.
S30
The structural integrity of the protease 1ST3 in the presence of PAA and γ-PGA
complex from Circular dichroism (CD) spectroscopy
The structural integrity of 6x10-5 mol L-1 protease 1ST3 in the absence and presence
of 2.2x10-3 mol L-1 PAA or 0.66x10-4 mol L-1 γ-PGA was analyzed by using CD
spectroscopy. The Far-UV CD spectra in Figure S13 A show that the protease 1ST3
has two single minimum around 208 nm and 222 nm and a stronger positive at ~192
nm, typical for α-helical proteins like serine proteases.3, 26 In the presence of both
polymers, the structure of the protease 1ST3 is not changed as shown in Figure S13
B.
Figure S13. Normalized Far-UV CD spectra of 6x10-5 mol L-1 native protease 1ST3 in A:
15°dGH water and B: in 15°dGH water containing 2.2x10-3 mol L-1 PAA (red line) or 0.66x10-
4 mol L-1 γ-PGA (blue line).
200 210 220 230 240 250 260
-30
-20
-10
0
10
20
30
40
50
60
Protease
[m
de
g]
Wavelength [nm]
200 210 220 230 240 250 260
-30
-20
-10
0
10
20
30
40
50
60
Protease
Protease + PAA
Protease + -PGA
A B
S31
Figure S14: protease 1ST3 and PAA for 3 independent production runs of MD simulation
(A) RMSF of aminoacid residues
(B) Contact area between protease 1ST3 and PAA
(C) interaction energy (Columbic and LJ energy)
(D) number of hydrogen bond between protease 1ST3 and PAA
A B
C D
S32
Figure S15: protease 1ST3 and PAA for 3 independent production runs of MD simulation
(A) binding free energy between protease 1ST3 and PAA
(B) Contribution of a particular residue to the binding free energy
(C) Contact map of aminoacids residue during simulation
(D) Number of contacts (distance < 0.35 nm) between protease 1ST3 and PAA
A B
C D
S33
Figure S16: protease 1ST3 and γ-PGA for 3 independent production runs of MD simulation
(A) RMSF of aminoacid residues
(B) Contact area between protease 1ST3 and γ-PGA
(C) interaction energy (Columbic and LJ energy)
(D) number of hydrogen bond between protease 1ST3 and γ-PGA
A B
C D
S34
Figure S17: protease 1ST3 and γ-PGA for 3 independent production runs of MD simulation
(A) binding free energy between protease 1ST3 and γ-PGA
(B) Contribution of a particular residue to the binding free energy
(C) Contact map of aminoacids residue during simulation
(D) Number of contacts (distance < 0.35 nm) between protease 1ST3 and γ-PGA
A B
C D
S35
Effect of ionic strength on protease 1ST3 bossting in the presence of
polyelectrolytes PAA and γ-PGA.
Varied concentrations of ionic strength were investigated on the protease boosting in
presence of PAA or γ-PGA in water by using Suc-AAPF-pNA assay (Figure S18 A-
D). To adjust the ionic strength in the water system, CaCl2·2H2O was used to mimic
the water system, which was used to quantify the protease boosting effects. The
boosted protease activity in the presence of 1.1x10-4 mol L-1 PAA is not affected by
increasing ionic strength (Figure S18 A and B). In the presence of 3.3x10-6 mol L-1 γ-
PGA, the proteolytic activity and the resulting boosting is decreased by gradually
increased ionic strength (Figure S18 C and D).
Figure S18: Proteolytic activity in the presence of the polyelectrolytes PAA and γ-PGA
at varied ionic strength. Proteolytic activity (slope min-1) of protease 1ST3 supernatant (1:5
dilution, filled black circle) in the presence of A: 1.1x10-4 mol L-1 PAA (filled blue circle) or C:
3.3x10-6 mol L-1 γ-PGA (filled red circle) at ionic strength of 5.6, 5.9, 8.6, 11.6, 20.6 and 35.6
mmol L-1 by using the Suc-AAPF-pNA assay across 10 min incubation. Boosting of protease
1ST3 by B: PAA and D: γ-PGA in dependence of ionic strength.
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.01.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
Protease
Protease + -PGA
Pro
tease p
erf
orm
an
ce
tow
ard
Su
c-A
AP
F-p
NA
[Slo
pe
min
-1]
Ionic strength [mmol L-1]
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.01.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
Protease
Protease + PAA
Pro
tea
se p
erf
orm
an
ce
tow
ard
Su
c-A
AP
F-p
NA
[Slo
pe
min
-1]
Ionic strenght [mmol L-1]
CA
5 10 15 20 25 30 35 401.0
1.1
1.2
1.3
1.4
1.5
B
oo
sti
ng
Ionic strength [mmol L-1]
5 10 15 20 25 30 35 401.0
1.1
1.2
1.3
1.4
1.5
B
oo
sti
ng
Ionic strength [mmol L-1]
B D
Ionic strength [mmol L-1]
S36
MD study of interaction of protease 1ST3 and two polyelectrolyte chains
MD simulations indicated that the protease has two favorable polyelectrolyte binding
regions, specifically surface amino acid residues in the close proximity of Ca2+
binding sites (i.e. Ca-1, Ca-2). Even though each Ca2+ binding sites theoretically can
attract a polyelectrolyte chain, MD simulations with more number of polymer chains
(PAA or γ-PGA) did not show simultaneous binding of two polymer chains to protease
(Figure S19). This might be due to the lack of attractive force after binding of the one
polymer chain to the protease surface. Comparison of protease-polymer interface
shown in Figure S14 and S16, showed that upon binding γ-PGA polyelectrolyte
chain covers a larger portion of the protease surface than PAA.
Figure S19: Number of contacts between PAA chains (chain 1 and chain 2) and protease
1ST3 along MD simulation trajectory.
S37
Electrostatic potential distribution on protease surface upon binding of PAA
In order to evaluate the proposed mechanisms, we have calculated electrostatic
potential distribution on protease surface upon binding of PAA (Figure S20). As it
can be clearly seen in Figure S20, neutralization of the positive charged residues
especially close to the Ca2+ binding sites changes the surface charge and slightly the
polarity of binding cleft of the protease.
Figure S20: A 3D structure of protease 1ST3 depicted as cartoon highlighting substrate
binding cleft and Ca2+ ions (Ca-1 (magenta) and Ca-2 (green)). Catalytic triad (D32, H62,
S215) and the oxyanion hole (N153) are shown as red sticks. Electrostatic potential
distribution on protease surface B: Protease, C: protease-PAA complex. The potential
computed is shown from -2.0 kcal/mol/e (red) to +2.0 kcal/mol/e (blue). Two sides of the
protease are shown in order to provide a complete view of the surface. Each view in (B) and
(C) of the protease has the same orientation as in (A).
180°
Ca-1
Ca-2
Ca-2
Ca-1
PAA
PAA
A B C
S38
Primers used for site-saturation mutagenesis (SSM) and site-directed
mutagenesis (SDM) of the protease 1ST3 gene:
Table S4: Primers used for SSM of the protease 1ST3 gene in pHY300PKL.
Name Sequence (5‘ to 3‘)
Fw_SSM_S76_MT GCTGCTTTAAACAATNNKATTGGCGTTCTT
Rev_SSM_S76_MT AAGAACGCCAATMNN ATTGTTTAAAGCAGC
Fw_SSM_I77_MT TTAAACAATTCGNNKGGCGTTCTTGGC
Rev_SSM_I77_MT GCCAAGAACGCCMNNCGAATTGTTTAA
Fw_SSM_A166_MT CCGGCCCGTTATNNKAACGCAATGGCA
Rev_SSM_A166_MT TGCCATTGCGTTMNN ATAACGGGCCGG
Fw_SSM_A188_MT TCACAGTATGGCNNKGGGCTTGACATT
Rev_SSM_A188_MT AATGTCAAGCCCMNNGCCATACTGTGA
Fw_SSM_D191_MT GGCGCAGGGCTTNNKATTGTCGCACCA
Rev_SSM_D191_MT TGGTGCGACAATMNN AAGCCCTGCGCC
Fw_SSM_V238_MT TCTTGGTCCAATNNKCAAATCCGCAACC
Rev_SSM_V238_MT GGTTGCGGATTTGMN NATTGGACCAAG
Fw_SSM_Q239_MT TGGTCCAATGTANNKATCCGCAACCAT
Rev_SSM_Q239_MT ATGGTTGCGGATMNNTACATTGGACCA
Fw_SSM_N242_MT GTACAAATCCGCNNKCATCTAAAGAAT
Rev_SSM_N242_MT ATTCTTTAGATGMNNGCGGATTTGTAC
Fw_SSM_K245_MT CGCAACCATCTANNKAATACGGCAACG
Rev_SSM_K245_MT CGTTGCCGTATTMNNTAGATGGTTGCG
S39
Table S5: Primers used for SDM of the protease 1ST3 gene in pHKL.
Name Sequence (5‘ to 3‘)
Fw_SDM_S76W_MT GCTTTAAACAATTGGATTGGCGTTCTT
Rev_SDM_S76W_MT TACGCCAAGAACGCCAATCCAATTGTTTAAAGC
Fw_SDM_S76H_MT GCTTTAAACAATCATATTGGCGTTCTT
Rev_SDM_S76H_MT TACGCCAAGAACGCCAATATGATTGTTTAAAGC
Fw_SDM_I77Q_MT TTAAACAATTCGCAGGGCGTTCTTGGC
Rev_SDM_I77Q_MT CGC TAC GCC AAG AAC GCC CTG CGA ATT GTT TAA
Fw_SDM_A188L_MT TCACAGTATGGCTTGGGGCTTGACATT
Rev_SDM_A188L_MT TGCGACAATGTCAAGCCCCAAGCCATACTGTGA
Fw_SDM_V238L_MT TCTTGGTCCAATTTGCAAATCCGCAACC
Rev_SDM_V238L_MT TAGATGGTTGCGGATTTGCAAATTGGACCAAGA
Fw_SDM_V238A_MT TCTTGGTCCAATGCTCAAATCCGCAAC
Rev_SDM_V238A_MT TAGATGGTTGCGGATTTGAGCATTGGACCAAGA
Fw_SDM_V238R_MT TCTTGGTCCAATCGGCAAATCCGCAACC
Rev_SDM_V238R_MT TAGATGGTTGCGGATTTGCCGATTGGACCAAGA
Fw_SDM_V238S_MT TCTTGGTCCAATTCTCAAATCCGCAACC
Rev_SDM_V238S_MT TAGATGGTTGCGGATTTGAGAATTGGACCA AGA
Fw_SDM_V238W_MT TCTTGGTCCAATTGGCAAATCCGCAAC
Rev_SDM_V238W_MT TAGATGGTTGCG GATTTGCCAATTGGACCAAGA
Fw_SDM_V238P_MT TCTTGGTCCAATCCGCAAATCCGCAAC
Rev_SDM_V238P_MT TAGATGGTTGCG GATTTGCGGATTGGACCAAGA
Fw_SDM_N242W_MT GTACAAATCCGCTGGCATCTAAAGAAT
Rev_SDM_N242W_MT TGCCGTATTCTT TAGATGCCAGCGGATTTGTAC
Fw_SDM_N242G_MT GTACAAATCCGCGGGCATCTAAAGAAT
Rev_SDM_N242G_MT TGCCGTATTCTTTAGATGCCCGCGGATTTGTAC
Fw_SDM_K245R_MT CGCAACCATCTAAGGAATACGGCAACG
Rev_SDM_K245R_MT TAAGCTCGTTGCCGTATTCCTTAGATGGTTGCG
Fw_SDM_K245N_MT CGCAACCATCTAAATAATACGGCAACG
Rev_SDM_K245N_MT TAAGCTCGTTGCCGTATTATT TAGATGGTTGCG
Fw_SDM_S215Y_MT TTAAACGGTACATACATGGCTACTCCT
Rev_SDM_S215Y_MT AACATGAGGAGTAGCCATGTATGTACCGTTTAA
S40
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