High-Yielde. Coli-Based Cell-Free Expression Ofhuman Proteins

High-yieldE. coli-based cell-free expression ofhuman proteins

Erich Michel1,Kurt Wüthrich1*

1Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.

*Corresponding author:

Supplementary Material

Figure captions

Figure S1:Comparison of soluble protein yields obtained permL reaction mixture when expressing the four target proteins ppiB, FABP4, SF20 and MTHFS without or with the N-terminal GB1-domain in the vectors pIVEX2.4 and pCFX1.

Table S1Oligonucleotide primers for cloning of the target proteins
Protein (UniGene Number) / 5‘- and 3‘-Oligonucleotide Primer
MARCKS-related protein (Hs.75061) / GGAATTCCATATGGGCAGCCAGAGCTCC
CGCGGATCCTTACTCATTCTGCTCAGCGCTG
Purkinje cell protein 2 homolog (PCP2H)
(Hs.591400) / GGAATTCCATATGATGGATCAGGAGGAGAAGAC
CGCGGATCCTCAGGGGGCTTGTGTCGG
Methenyl-THF synthetase (MTHFS) / GGAATTCCATATGGCGGCGGCAGCGG
CGCGGATCCTTAAGCTGTTGACGAGTCTTCG
(Hs.459049)
-interferon-inducible lysosomalthiolreductase
(GILT), (Hs.14623) / GGAATTCCATATGAATGCACCGCTTGTCAATGTG
CGCGGATCCTTACTTGCCCTGGTACAACTGG
Mitochondrial Methyl-malonyl-CoAepimerase
(MMCE), (Hs.94949) / GGAATTCCATATGCAAGTGACAGGTTCTGTGTGG
CGCGGATCCTCAAGCTTGCTCCAGTTCCAC
Stromal cell-derived growth factor SF20 (SF20) (Hs.465645) / GGAATTCCATATGGTGTCCGAGCCCACGAC
CGCGGATCCTCACAGCTCAGTGCGCGATG
Adipocyte fatty acid-binding protein 4 (FABP4) (Hs.391561) / GGAATTCCATATGTGTGATGCTTTTGTAGGTAC
CGCGGATCCTTATGCTCTCTCATAAACTCTCG
Peptidyl-prolyl cis-transisomerase B (ppiB) / GGAATTCCATATGGATGAGAAGAAGAAGGGGCC
CGCGGATCCTTACTCCTTGGCGATGGCAAAG
(Hs.434937)
Complexin-1 (Hs.478930) / GGAATTCCATATGGAGTTTGTGATGAAGCAGG
CGCGGATCCTTACTTCTTGAGCATGTCCTGCAG
Dihydrofolatereductase (DHFR) / GGAATTCCATATGGTTGGTTCGCTAAACTGC
CGCGGATCCTTAATCATTCTTCTCATATACTTCAAATTT
(Hs.592364)
Table S2 Composition of the cell-free reaction mixture
Component (concentration) / Volume
ERCN-Solution
DEPC-treated water
tRNA (17.5 mg/mL)
NaN3 (0.77 M)
Mg(OAc)2 (1.6 M)
Amino acid mix (20 mM)
Creatinekinase (87 M)
T7 RNA polymerase (65 M)
S30 extract (400 A260 Units)
Plasmid DNA (1 mg/mL) / 3.73 mL
1.31 mL
100 L
100 L
43L
750 L
667 L
100 L
3.00 mL
200 L
Total volume / 10 mL
Table S3 Composition of the energy-regeneration-cofactor-nucleotide solution (ERCN)
Component (concentration) / Volume
HEPES-KOH, pH 7.5 (1 M)
PEG-8000 (40% (w/v))
KOAc (6 M)
DTT (0.55 M)
ATP, pH 7.0 (400 mM)
GTP, pH 7.0 (400 mM)
CTP, pH 7.0 (400 mM)
UTP, pH 7.0 (400 mM)
Folinic acid.Ca2+ (5.27 mM)
cAMP.Na+ (100 mM)
NH4OAc (2.2 M)
Creatine phosphate (1 M)
DEPC-treated water / 1.55mL
2.71mL
890 L
87L
81L
58L
58L
58L
345 L
173L
330L
2.16 mL
1.5 mL
Total volume / 10 mL
Table S4 Composition of the feeding solution used in continuous-exchange cell-free protein expression
Component (concentration) / Volume
ERCN-Solution
DEPC-treated water
NaN3 (0.769 mM)
S30 buffer
Amino acid mix (20 mM)
Mg(OAc)2 (1.6 M) / 37.3mL
23.8mL
1mL
30mL
7.5 mL
0.43 mL
Total volume / 100 mL
Table S5 Protein yields from cell-free expression
Protein / Protein purified from 10 mL reaction mixture as a fusion construct with GB1
MARCKS-related protein / 8.6 mg
Purkinje cell protein 2 homolog / 2.4 mg
Methenyl-THF synthetase / 2.7 mg
-interferon-inducible
lysosomalthiolreductase / 4.6 mg
Mitochondrial methylmalonyl-
CoAepimerase / 1.8 mg
Stromal cell-derived growth
factor SF20 (C19orf10) / 3.2 mg
Adipocyte fatty acid-
binding protein 4 / 5.4 mg
Peptidyl-prolyl cis-trans
isomerase B / 5.3 mg
Complexin-1 / 8.4 mg
Dihydrofolatereductase / 4.5 mg
Table S6 Expense for chemicals used to prepare 15 mL of S30 cell extract
Component / Quantity / Expense
[CHF]*
9 L culture medium:
K2HPO4
KH2PO4
Glucose
Yeast extract / 130 g
50 g
90 g
90 g / 8
2
1
12
4 L S30 buffer:
KOAc
Mg(OAc)2
Trizma base
DTT
-Mercaptoethanol
DEPC / 24 g
12 g
5 g
617 mg
2 mL
4 mL / 2
1

4

17
Preincubation components:
Trizma base
Mg(OAc)2
ATP
PEP
DTT
Amino acid mix
Pyruvatekinase / 213 mg
12 mg
48 mg
104 mg
4 mg
1 mg
40 U / 


23



Total for 15 mL of S30 extract: 70
*  stands for “less than 1 CHF”
Table S7 Expense for chemicals used to prepare the reaction mixture for 10 mL of batch-mode cell-free expression
Component / Batch Mode / CECF Mode
Quantity / Expense
[CHF]* / Quantity / Expense
[CHF]*
HEPES / 137 mg /  / 1.5 g / 2
E. colitRNA / 3.5 mg / 13 / 3.5 mg / 13
PEG-8000 / 325 mg /  / 3.6 g / 
ATP / 7.4 mg /  / 81 mg / 
GTP / 4.9 mg / 3 / 54 mg / 28
CTP / 4.9 mg / 1 / 53 mg / 14
UTP / 5.1 mg /  / 56 mg / 10
Folinic acid, Ca2+-salt / 0.4 mg /  / 4 mg / 4
cAMP, Na+-salt / 2.4 mg / 2 / 26 mg / 22
NH4OAc / 21 mg /  / 234 mg / 
Creatine phosphate / 264 mg / 7 / 2.9 g / 81
DTT / 2.7 mg /  / 35 mg / 
Mg(OAc)2 / 14 mg /  / 249 mg / 
Creatinekinase / 2.5 mg / 3 / 2.5 mg / 3
NaN3 / 5 mg /  / 55 mg / 
KOAc / 195 mg /  / 2.3 g / 
Unlabeled amino acids / 60 mg / 7 / 660 mg / 75
15N-amino acids / 60 mg / 75 / 660 mg / 827
13C,15N-amino acids / 60 mg / 133 / 660 mg / 1465
S30 Extract / 3 mL / 15 / 3 mL / 15
*  stands for “less than 1 CHF”