Supplemental Material for
Development of a full-length human protein production pipeline
Justin Saul1, Brianne Petritis1, Sujay Sau2, Femina Rauf1, Michael Gaskin1, Benjamin OberReynolds1, Irina Mineyev3, Mitch Magee1, John Chaput2, Ji Qiu1*, Joshua LaBaer1*
1
Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State
University, PO Box 876401, Tempe, AZ 85287-6401, USA.
2
Center for Evolutionary Medicine and Informatics, Biodesign Institute, Arizona State
University, Tempe, AZ 85287-5301, USA.
3
PerkinElmer, Inc., Hopkinton, MA 01748, USA
*To whom correspondence should be addressed. Email: [email protected];
[email protected]
This file includes:
Figures S1-S5
Table S1
Figure S1. Cloning E. coli, WGE, and HCE destination vectors. (A) Design of expression vectors for all
three expression systems. The pCPD E coli vector contains a T7lac promoter, the pJSP6 WGE vector
contains an SP6 promoter, and the HCE pJFT7 vector contains a T7 promoter. (B thru D) Cloning
outline of the three empty (E) vectors, for use as controls, and the Gateway destination vectors
containing the Gateway death cassette (DC).
Table S1. Collection of full-length human proteins tested.
Target
Size
(kD)
class
localization
uniprot ID
GTF2H5
8
transcription factor
Nucleus.
Q6ZYL4
PHPT1
14
phosphatase
Cytoplasm.
Q9NRX4
CDKN2A
14
kinase modulator
Nucleus > nucleolus. Nucleus > nucleoplasm.
Q8N726
GSKIP*
16
signaling modulator
Q3TBR1
HSPB11
16
chaperone
Q9Y547
OMP
19
signaling modulator
Cytoplasm.
P47874
TPPP3
19
tubulin modulator
Cytoplasm > cytoskeleton.
Q9BW30
NIP7
20
rRNA processing
Nucleus > nucleolus.
Q9Y221
BCL2A1
20
signaling modulator
Cytoplasm.
Q16548
RBBP9
21
signaling modulator
O75884
HRAS
21
GTPase/signaling
Cytoplasm. Nucleus.
Cell membrane. Cell membrane; Lipid-anchor;
Cytoplasmic side. Golgi apparatus.
BCL10
26
signaling modulator
Cytoplasm > perinuclear region. Membrane raft.
O95999
CRISP1
28
Secreted.
P54107
CCND1
34
cyclin
C-JUN
36
transcription factor
Nucleus.
P05412
MAPK1
41
kinase
Nucleus. Cytoplasm > cytoskeleton > centrosome.
P28482
HADHSC
42
dehydrogenase
Mitochondrion matrix.
Q16836
P53
45
tumor suppressor
Cytoplasm. Nucleus. Nucleus > PML body. ER.
P04637
SNIP1
46
transcription factor
Nucleus.
Q8TAD8
GSK3B
47
kinase
Cytoplasm. Nucleus. Cell membrane.
P49841
IKBKG
48
signaling modulator
Cytoplasm. Nucleus.
Q9Y6K9
C-MYC
51
transcription factor
P01106
MYOT
55
actin modulator
AKT3
56
kinase
C-SRC
50
kinase
NF2
70
signaling modulator
PRKCB
77
kinase
Nucleus > nucleoplasm. Nucleus > nucleolus.
Cell membrane > sarcolemma. Cytoplasm >
cytoskeleton.
Nucleus. Cytoplasm. Membrane; Peripheral membrane
protein.
Cell membrane. Mitochondrion inner membrane.
Nucleus. Cytoplasm > cytoskeleton.
Cell projection > filopodium membrane; Peripheral
membrane protein; Cytoplasmic side.
Cytoplasm. Nucleus. Membrane; Peripheral membrane
protein.
CTNNB1
85
signaling modulator
Cytoplasm. Nucleus. Cytoplasm > cytoskeleton.
P35222
MALT1
92
signaling modulator
Cytoplasm > perinuclear region. Nucleus.
Q9UDY8
PI3KCG
97
kinase
P48736
ABL1
123
kinase
Cytoplasm. Cell membrane.
Cytoplasm > cytoskeleton. Nucleus. Mitochondrion.
Nucleus membrane; Lipid-anchor.
P01112
Q6FI00
*Mouse homolog of human protein UFC1
Q9UBF9
Q9Y243
P12931
P35240
P05771
P00519
Figure S2. E. coli, wheat germ extract (WGE), and HeLa cell extract (HCE) high throughput expression
formats. The volume of each reaction is 2 mL culture for E. coli, 226 µL lysate for WGE, and 100 µL
lysate for HCE. Expression is performed in automation-compatible 24 or 96 well formats. WGE and HCE
systems utilize continuous exchange cell free (CECF) designs to increase translation efficiency.
Figure S3. Comparison of SDS-PAGE gel and LabChip virtual gel analysis of HaloTagged full-length
proteins expressed in HCE. (A) Coomassie staining of 1 uL soluble fraction of translation reaction
volume. (B) Fluorescent image of the same gel prior to coomassie staining. (C) LabChip virtual gel of the
same collection of samples. A 1:5 dilution of HCE reaction was incubated with 4 uM HaloTag Alexa660
ligand for 20 min in a 2:1 sample:ligand ratio prior to subsequent dilution in Laemmli or LabChip sample
buffer and denaturation.
Figure S4. Plot of LabChip SIFT signal and slab gel quantification for the protein collection expressed in
HCE.