Supplementary Materials for:
A simplified and robust protocol for immunoglobulin expression in
Escherichia coli cell-free protein synthesis systems
Qi Cai+, Jeffrey A. Hanson+, Alexander R. Steiner, Cuong Tran, Mary Rose Masikat, Rishard
Chen, James F. Zawada, Aaron K. Sato, Trevor J. Hallam, and Gang Yin*
Sutro Biopharma, Inc. 310 Utah Avenue, Suite 150, South San Francisco, CA 94080
+
These authors contributed equally to this work.
* Correspondence to: Gang Yin; Email: gyin@sutrobio.com
1
Contents
Stock solutions for cell-free reactions................................................................................................ 2
Table S1: Detailed cost calculations .................................................................................................. 3
Figure S1: Representative 14C gel for cell-free IgG quantification................................................. 5
Figure S2: Time course of Trastuzumab expression using conventional and simplified
protocols ............................................................................................................................................... 6
Stock solutions for cell-free reactions
Though most of the reaction components can be added directly in powdered form to the master mix
solution, for cell-free reactions described in the manuscript, stock solutions of each compound were made
for the present work to facilitate titration of the individual reaction components.
The amino acid tyrosine was prepared at 50 mM at pH 11 using KOH due to its low solubility at neutral
pH. A 25x amino acid stock solution containing 50 mM each of the remaining 19 amino acids was
prepared and had a pH of 5.2. A 50x nucleotide stock solution was created with 60 mM ADP and 43 mM
each of GMP, UMP and CMP without adjustment to the pH. A 1 M phosphate solution was prepared at
pH 7 with 615 mM K2HPO4 and 385 mM KH2PO4.
The following stock solutions were made in milliQ water without adjustments to the pH: 2 M sodium
pyruvate, 5 M potassium glutamate, 500 mM magnesieum glutamate, 1 M ammonium glutamate, 33 mM
NAD, 80 mM coenzyme A, 1 M potassium oxalate, 3.4 mg/mL folinic acid, 100 mM oxidized glutathione
(GSSG), 80 mM hemimagnesium glutamate, 200 mM spermidine, and 200 mM putrescine.
Total E. coli tRNA was resuspended at 17 mg/mL. T7 RNA polymerase was produced in house and
stored in 50 mM Tris (pH 7.8) 10% sucrose, 300 mM NaCl, 2 mM DTT and 0.1 mM EDTA at -80 °C at
concentrations of 5-10 mg/mL. Plasmid DNA was prepared by maxiprep (Qiagen) and resuspended in
water at concentrations of 0.2 or 1 mg/mL.
The pH of the final master mix solution is 6.7.
2
Table S1: Detailed cost calculations
Conventional protocol
Reagent
Folinic acid
E. coli total tRNA
Vendor
catalog #
Sigma
47612-1G
Roche Diagnostics
10109550001
unit
price
1g
572
0.5g
587
M.W.
Conc
0.034g/L
$/L
%
Conc
$/L
%
19.45
2.94
0g/L
0.00
0.00
0mg/L
0.00
0.00
0.00
0.00
0.00
0.00
0.67
1.60
0.17mg/L 199.58 30.17
Transription
Simplified protocol
219.03 33.11
L-Glutamic Acid, Ammonium Salt
L-Glutamic acid hemimagnesium salt tetrahydrate
MP Biomedicals
02180595.1-100g
100g
77
164.2
10mM
1.26
0.19
Sigma
49605-250G
L-Glutamic acid monopotassium salt monohydrate
Potassium oxalate monohydrate
Sigma
G1501-1KG
250g
53.9
1000g
243.5
Sigma
223425
12000g
Sodium pyruvate
Sigma
P2256
β-Nicotinamide adenine dinucleotide hydrate
Sigma
Coenzyme A sodium salt hydrate
388.61
8mM
0.67
0.10
8mM
203.23
130mM
6.43
0.97
260mM
681
184.23
4mM
0.04
0.01
4mM
0.04
0.10
500g
789.5
110.04
33mM
5.73
0.87
0mM
0.00
0.00
N1636
1g
426.5
663.43
0.33mM
93.37
14.11
0mM
0.00
0.00
Sigma
C3144-1G
1g
1480
767.53
0.27mM
306.70 46.36
0mM
0.00
0.00
K2HPO4·3H20
Sigma
P5504
10000g
879
228.23
9.2mM
0.18
0.03
9.2mM
0.18
0.44
KH2PO4
Sigma
P5379
10000g
454.5
136.09
5.8mM
0.04
0.01
5.8mM
0.04
0.09
Energy-related
0mM
414.44 62.65
12.87 30.76
13.80 32.99
Putrescine
MP Biomedicals
0210044180-100g
100g
63.35
88.15
1mM
0.06
0.01
0mM
0.00
0.00
Spermidine
Sigma
S2626
25g
466
145.25
1.5mM
4.06
0.61
1.5mM
4.06
9.71
4.12
0.62
4.06
9.71
4.68
0.71
4.68
11.19
4.68
0.71
4.68
11.19
Polyamines
L-Glutathione oxidized , GSSG
Sigma
G4376
1000g
3820
612.6
2mM
Folding
2mM
AMP
GMP
Sigma
A1752
25g
210.5
347.22
1.2mM
3.51
0.53
1mM
3.51
8.39
Sigma
G8377
100g
790
407.18
0.86mM
2.77
0.42
1mM
2.77
6.61
UMP
Sigma
U6375
10g
135.5
369.15
0.86mM
4.30
0.65
1mM
4.30
10.28
CMP
Sigma
C1006
5g
103
367.16
0.86mM
6.50
0.98
1mM
6.50
15.55
L-Alanine
Sigma
A7627
1000g
450.5
89.09
2mM
0.08
0.01
2mM
0.08
0.19
L-Arginine
Sigma
A5006
1000g
219
174.2
2mM
0.08
0.01
2mM
0.08
0.18
L-Asparagine
Sigma
A0884
1000g
384.5
132.12
2mM
0.10
0.02
2mM
0.10
0.24
L-Aspartic Acid
Sigma
A7219
1000g
203
133.1
2mM
0.05
0.01
2mM
0.05
0.13
3
L-Cysteine
Sigma
C7352
1000g
601
121.16
2mM
0.15
0.02
2mM
0.15
0.35
L-Glutamine
Sigma
G8540
1000g
445
146.15
2mM
0.13
0.02
2mM
0.13
0.31
Glycine
Sigma
G7126
25000g
914
75.07
2mM
0.01
0.00
2mM
0.01
0.01
L-Histidine
Sigma
H8000
1000g
552
155.16
2mM
0.17
0.03
2mM
0.17
0.41
L-Isoleucine
Sigma
I2752
1000g
742
131.18
2mM
0.19
0.03
2mM
0.19
0.47
L-Leucine
Sigma
L8000
1000g
411
131.18
2mM
0.11
0.02
2mM
0.11
0.26
L-Lysine·H20
Sigma
L7039
25000g
3005
182.65
2mM
0.04
0.01
2mM
0.04
0.10
L-Methionine
Sigma
M9625
1000g
274
149.21
2mM
0.08
0.01
2mM
0.08
0.20
L-Phenylalanine
Sigma
P2126
1000g
425.5
165.190
2mM
0.14
0.02
2mM
0.14
0.34
L-Proline
Sigma
P0380
5000g
2180
115.13
2mM
0.10
0.02
2mM
0.10
0.24
L-Serine
Sigma
S4311
1000g
799
105.09
2mM
0.17
0.03
2mM
0.17
0.40
L-Threonine
Sigma
T8625
1000g
818
119.12
2mM
0.19
0.03
2mM
0.19
0.47
L-Tryptophan
Sigma
T0254
1000g
573
204.23
2mM
0.23
0.04
2mM
0.23
0.56
L-Tyrsosine
Sigma
T8566
1000g
506
181.190
1mM
0.09
0.01
1mM
0.09
0.22
L-Valine
Sigma
V0500
1000g
382.5
117.15
2mM
0.09
0.01
2mM
0.09
0.21
Building Blocks
19.29
2.92
Total
661.56
19.29 46.12
41.83
4
Figure S1: Representative 14C gel for cell-free IgG quantification
Figure S1: Representative 14C gels used to calculate assembled IgG yields. These gels show the titration
of sodium pyruvate at two potassium glutamate concentrations. This gel was used to calculate
trastuzumab concentrations in Figure 4b in the main text.
5
Figure S2: Time course of trastuzumab expression using conventional and simplified
protocols.
Figure S2. Time course of trastuzumab expression in conventional and simplified protocols showing that
the kinetics of expression are similar in the two systems. Expression was carried out on 100 mL scale in a
DASGIP DASbox mini-bioreactor. Reactions were performed in triplicate. Protein expression was
quantified by purifying 0.6 mL of each cell free reaction using a 40 µL protein A phytips (Phynexus) on a
BioMek FX (Beckman Coulter) liquid handler. The final recovery yields were about 350 mg/L and
protein recovery was estimated to be 50%.
6