Download Supplementary Information Dissecting limiting factors of the Protein

Supplementary Information
Dissecting limiting factors of the Protein synthesis Using Recombinant Elements
(PURE) system
Jun Li1,2,*, Chi Zhang1, Poyi Huang1, Erkin Kuru1, Eliot T. C. Forster-Benson3, Taibo Li4 and
George M. Church1,2,*
1. Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2. Wyss Harvard Institute of Biologically Inspired Engineering, 3 Blackfan Circle, Boston,
MA 02115, USA.
3. Ravenwood High School, 1724 Wilson Pike, Brentwood, TN 37027, USA.
4. Department of Electrical Engineering and Computer Science, Massachusetts of Institute
of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
* Correspondences should be addressed to Jun Li at [email protected] or George M.
Church at [email protected].
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Supplementary Figure 1. Time course study of PURE system protein synthesis with three model proteins.
A. Assessment of PURE system translation by production of functional TC-Fluc. Equal aliquots of PURE
reactions were taken at different time points and TC-Fluc activity was measured by luciferase assay.
Actual amount of TC-Fluc produced was calculated using a pre-measured standard curve. Values
represent averages and error bars are ± standard deviations, with n=3. B. Assessment of total PURE
system translation yields by production of HaloTag fusion proteins HT-Ch-TolA and HT-EntF-Ch-TolA.
Equal aliquots of PURE reactions were taken at different time points and incubated with HaloTag TMR
Ligand. Samples were run on a SDS-PAGE and scanned by a typhoon scanner with filter set
(555nmEx/580nmEm). HT: HaloTag; Ch: mCherry. TolA is a C-terminal 171-amino-acid alpha-helical
spacer excised from E. coli TolA domain II. EntF is a multidomain enzyme from E. coli enterobactin
biosynthetic pathway. Asterisks * indicate full length HT-Ch-TolA and HT-EntF-Ch-TolA produced.
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Supplementary Figure 2. Inorganic phosphate (Pi) accumulation during PURE system proteins synthesis.
Pi concentrations were measured as described in methods section and are shown over the course of
batch PURE system reactions. Values represent averages and error bars are ± standard deviations, with
n=3.
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Supplementary Figure 3. Replenishing PURE system protein synthesis with Mg 2+ or Mg2+ and creatine
phosphate in combination at different time points. PURE system reactions synthesizing TC-Fluc were set
up and incubated at 37°C. Small molecule substrates as indicated were fed to the reaction at 15 min, 30
min, 45 min, 60 min or 75 min. Control reactions were equivalent volumes of water fed at 0 min. Overall
TC-Fluc produced was assessed by luciferase assay at 90 min. Values represent averages and error bars
are ± standard deviations, with n=3. PURE system reaction feed with A. 4.8 mM magnesium acetate; and
B. 20 mM creatine phosphate and 4.8 mM magnesium acetate in combination.
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Supplementary Figure 4. Replenishing PURE reactions of TC-Fluc with 3 concentration sets of a
combination of DTT, 10-CHO-THF, creatine phosphate, magnesium acetate, AA and tRNA at 75 min.
Control reactions were fed equivalent volumes of water at 75 min. Reactions were carried out at 37 °C
for 3 h. Equal volumes of aliquots were taken at 75 min, 90 min, 120 min, 150 min and 180 min for
luciferase assay to measure the actual amount of TC-Fluc produced. Values represent averages and error
bars are ± standard deviations, with n=3.
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Supplementary Figure 5. TC-Fluc synthesis in the PURE system with different EF-P concentrations. PURE
system was assembled with plasmid encoding TC-Fluc in the presence of different concentrations of EF-P
protein as indicated. TC-Fluc activity was measured after 2 h incubation at 37°C. Values represent the
result from a single test.
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Supplementary Figure 6. Supplementing PURE system with EF4 in the presence of additional 7mM Mg2+.
A. EF4 effects on Fluc synthesis in the PURE system in the presence of additional 7mM Mg2+
concentration. Addition of various amounts of EF4. One aliquot of the reaction mixture was measured
with luciferase assay. Values represent averages and error bars are ± standard deviations, with n=3. B.
Effects of EF4 on HT-AcGFP synthesis in the PURE system with increased Mg2+ concentration. Sample
aliquot was incubated with HaloTag TMR Ligand, seperated on a SDS-PAGE and then scanned by a
typhoon scanner with filter set (555nmEx/580nmEm).
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Supplementary Figure 7. Effect of PrfH presence to the PURE system TC-Fluc synthesis. PURE system
was assembled with different templates encoding TC-Fluc as indicated, with or without 1.8 M PrfH
protein. TC-Fluc activity was measured after 2 h incubation at 37°C. Values represent averages and error
bars are ± standard deviations, with n=3. (N.S: not significant)
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Supplementary Figure 8. Assessment of full length and truncated products produced by the PURE
system when linear or circular templates were added (sc indicates stop codon). Reactions were
incubated at 37°C for 2 h and labelled using FIAsH-EDT2. Samples were then run on a SDS-PAGE and
scanned by a typhoon scanner with filter set (508nmEx/528nmEm).
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