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: u.schwaneberg@biotec.rWTh-aachen.de

,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

References

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