Supporting Information The Effect of ELP Sequence and Fusion ...
Supporting Information
The Effect of ELP Sequence and Fusion Protein Design on Concentrated Solution Self-Assembly Guokui Qin, Paola M. Perez, Carolyn E. Mills, and Bradley D. Olsen* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
Biosynthesis of mCherry-ELP fusion proteins Model globular proteins fused with coil-like proteins were synthesized using the red fluorescent protein mCherry and the thermoresponsive structural polymer ELPs, as described previously. The high-yield expression and purification of the model protein mCherry has been well-established, and its fluorescent nature provides a simple and robust spectrophotometric method for fusion characterization. Two ELP blocks, ELP0 and ELP1, with different amino acid sequences, were chosen and fused with mCherry to generate two series of mCherry-ELP fusions including mCherry-ELP0 and mCherryELP1. ELP1 (VPGVGVPGGGVPGAG(VPGVG)3VPGGGVPGAGVPGGGVPGVG)4, a new ELP chain, was designed with more hydrophilic properties for comparison with the previous ELP chain, ELP0 ((VPGVG)2IPGVG(VPGVG)2)20, renamed from the previous E20 chain. The new fusion ELP0a-mCherry was generated by adding 6XHis tag onto the N termini of ELP0-mCherry, while the mCherry-ELP1a was produced by removing 6XHis tag from the N termini of mCherry-ELP1. Compared to original mCherry-ELP0
fusion, the ELP chain size in fusion ELP0b-mCherry is smaller. Compared to the mCherry-ELP1 fusion, mCherry-ELP1b has a longer ELP chain. The position of the ELP block in the fusion was also changed to obtain the new fusion mCherry-ELP0c and ELP1c-mCherry by switching N/C termini of the ELP0a-mCherry and mCherry-ELP1 fusions, respectively.
Plasmid construction. The gene encoding for mCherry was prepared for insertion into the bacterial T7 promoter expression vector pET28a (Novagen EMD Chemicals, Inc. CA) by partial digestion with BamHI and HindIII and ligated to form a new plasmid pET28a/mCherry. The NheI/SpeI fragment of different ELP blocks was then designed and inserted at different positions in the corresponding sites of pET28a/mCherry, resulting
ELP
gene-based
8
different
plasmids,
pET28a/ELP0-mCherry
and
pET28a/mCherry-ELP1 for the production of the 8 different ELP0-based and ELP1based fusions, respectively. All of the recombinant expression plasmids were isolated from E. coli NEB5α cells (NEB, MA) with selection from Kanamycin resistance (50 µg/mL), and the correct constructs were verified by DNA sequencing. The full amino acid and DNA sequences of the fusion proteins are included in Figures S1 – S8.
Protein expression. All expression vectors were then transformed into E. coli tuner cells (BL21-DE3; NEB, MA). Authenticity of the clones was confirmed by DNA sequencing. A total of 5 mL of overnight cultures were grown in LB medium with 50 µg/mL kanamycin at 37 °C in a rotary shaker. These starter cultures were used for inoculation of 1 L of LB cultures with 50 µg/mL kanamycin at a ratio of 1/100 of starter to culture
volume. The expression was further performed at 20 °C for 48 h without induction untill bright pink proteins were observed, and the bacteria were harvested by centrifugation (10,000 g for 20 min at 4 °C). Pellets were stored at -80 °C.
Protein purification. The cell pellets were thawed and resuspended in lysis buffer (MENT buffer: 100 mM NaCl, 10 mM Tris, 1 mM EDTA, 5 mM MgCl2, pH 8.0), incubated with 1 mg/mL lysozyme at 4 oC for 30 min, and sonicated. The lysate was clarified, then thermally cycled between 37 °C and 4 °C three times, and further purified by FPLC under native conditions to preserve the chromophore bond of mCherry. Elution fractions containing purified, bright pink proteins were dialyzed into 20 mM Tris-Cl buffer, pH 8. The purity of the protein was confirmed by denaturing gel electrophoresis (SDS-PAGE) for both mCherry-ELP versions with a yield of ~ 50 mg/L.
MGWGSASGLVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGETTSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEI KQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRH STGGMDELYK Figure S1. Conceptual amino acid sequence of ELP0-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASGLVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTSMVSKGEEDNMAIIKE FMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPS DGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQL PGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK Figure S2. Conceptual amino acid sequence of ELP0a-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASGLVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTSMTG GQQMGRGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLK VTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLK LKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKTS Figure S3. Conceptual amino acid sequence of ELP0b-mCherry. The region corresponding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASMVSKGEEDNMAIIKEFMRFKVH MEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPA DIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQ KKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVN IKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSGLVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTS Figure S4. Conceptual amino acid sequence of mCherry-ELP0c. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVSKGEEDNMAIIKEFMRFKVHME GSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPCDGPVMQK KTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNI KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSVPGVGVPGGGVPGAGVPGVGVPGV GVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGG GVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGG GVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTTS Figure S5. Conceptual amino acid sequence of mCherry-ELP1. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGWGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVT QDSSLQDGEFIYKVKLRGTNFPCDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKTSVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPG VGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPG AGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPG VGVPGVGVPGGGVPGAGVPGGGVPGVGTSS Figure S6. Conceptual amino acid sequence of mCherry-ELP1a. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVSKGEEDNMAIIKEFMRFKVHME GSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPCDGPVMQK KTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNI KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSVPGVGVPGGGVPGAGVPGVGVPGV GVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGG GVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGG GVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSVP GVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVP GAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVP GVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVP GGGVPGAGVPGGGVPGVGTSVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAG VPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVG VPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGG VPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSS Figure S7. Conceptual amino acid sequence of mCherry-ELP1b. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGV PGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGV PGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGV PGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSMTGGQQMGRGSMV SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG EFIYKVKLRGTNFPCDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDA EVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSS Figure S8. Conceptual amino acid sequence of ELP1c-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
Figure S9.Transmittance as a function of temperature in 1 mg/mL solution indicates the thermoresponsive transition of ELP-mCherry fusions (a) mCherry-ELP0, (b) mCherryELP0a, (c) mCherry-ELP0b, (d) ELP0c-mCherry, (e) mCherry-ELP1, (f) mCherryELP1a, (g) mCherry-ELP1b, and (h) ELP1c-mCherry, where high transmittance (below the transition temperature, Tt) indicates individual molecules dispersed in solution, and low transmittance (above Tt) indicates the formation of macrophase separated structures.
Figure S10. Results of turbidity measurements on fusion proteins samples at 50 wt% in water. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherryELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherry-ELP1b, (h) ELP1cmCherry.
Figure S11. Results of bireinfringence measurements on fusion proteins samples at 50 wt% in water. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherry-ELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherry-ELP1b, (h) ELP1c-mCherry.
Figure S12. SAXS as a function of temperature shows different phase behaviors of mCherry-ELP fusion proteins at 50 wt%. From bottom to top, the curves were taken at 10 o C, 15 oC, 20 oC, 25 oC, 30 oC, 35 oC, and 40 oC . (a) ELP0-mCherry, (b) ELP0amCherry, (c) ELP0b-mCherry, (d) mCherry-ELP0c, (e) mCherry-ELP1, (f) mCherryELP1a, (g) mCherryELP1b, and (h) mCherry ELP1c.
Figure S13. Fusion protein absorbance spectra before dehydration, in film, and after rehydration, demonstrating the preservation of mCherry fluorescence for different fusion designs. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherryELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherryELP1b, and (h) mCherry ELP1c.
Table S1. Summary of full width half maxima and domain spacings calculated based on fits to peaks in SAXS data for all fusion proteins at 10 oC, 20 oC and 40 oC Fusion ELP0-mCherry
ELP0a-mCherry
ELP0b-mCherry
mCherry-ELP0c
mCherry-ELP1
mCherry-ELP1a
mCherry-ELP1b
ELP1c-mCherry
Temperature (oC) 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40
Peak 1 d (nm) FWHM (nm-1) 28.11 ± 0.08 0.0301 ± 0.0006 28.14 ± 0.09 0.0279 ± 0.0006 24.78 ± 0.04 0.0203 ± 0.0004 24.54 ± 0.07 0.0156 ± 0.0006 25.26 ± 0.08 0.0223 ± 0.0007 18.08 ± 0.08 0.125 ± 0.001 18.16 ± 0.08 0.111 ± 0.001 18.83 ± 0.02 0.0199 ± 0.0003 23.58 ± 0.01 0.0114 ± 0.0001 23.19 ± 0.02 0.0150 ± 0.0002 17.2 ± 0.3 0.327 ± 0.005 16.8 ± 0.3 0.353 ± 0.005 16.5 ± 0.3 0.314 ± 0.006 40.2 ± 0.5 0.113 ± 0.002 40.5 ± 0.8 0.121 ± 0.003 40.3 ± 0.9 0.126 ± 0.003 16.7 ± 0.2 0.220 ± 0.004 16.6 ± 0.2 0.222 ± 0.004 16.8 ± 0.2 0.214 ± 0.004
Table S2. Molecular weights of proteins as measured by MALDI-TOF compared to calculated protein molecular weight based on sequence Fusion ELP0-mCherry ELP0a-mCherry ELP0b-mCherry mCherry-ELP0c mCherry-ELP1 mCherry-ELP1a mCherry-ELP1b ELP1c-mCherry
Molecular Weight Calculate by Sequence (kDa) 69.4 71.8 47.1 71.8 46.3 43.9 73.7 46.3
Molecular Weight Measured by MALDI-TOF (kDa) 69.0 72.1 49.3 72.0 47.1 40.9 72.2 46.7
The Effect of ELP Sequence and Fusion Protein Design on Concentrated Solution Self-Assembly Guokui Qin, Paola M. Perez, Carolyn E. Mills, and Bradley D. Olsen* Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
Biosynthesis of mCherry-ELP fusion proteins Model globular proteins fused with coil-like proteins were synthesized using the red fluorescent protein mCherry and the thermoresponsive structural polymer ELPs, as described previously. The high-yield expression and purification of the model protein mCherry has been well-established, and its fluorescent nature provides a simple and robust spectrophotometric method for fusion characterization. Two ELP blocks, ELP0 and ELP1, with different amino acid sequences, were chosen and fused with mCherry to generate two series of mCherry-ELP fusions including mCherry-ELP0 and mCherryELP1. ELP1 (VPGVGVPGGGVPGAG(VPGVG)3VPGGGVPGAGVPGGGVPGVG)4, a new ELP chain, was designed with more hydrophilic properties for comparison with the previous ELP chain, ELP0 ((VPGVG)2IPGVG(VPGVG)2)20, renamed from the previous E20 chain. The new fusion ELP0a-mCherry was generated by adding 6XHis tag onto the N termini of ELP0-mCherry, while the mCherry-ELP1a was produced by removing 6XHis tag from the N termini of mCherry-ELP1. Compared to original mCherry-ELP0
fusion, the ELP chain size in fusion ELP0b-mCherry is smaller. Compared to the mCherry-ELP1 fusion, mCherry-ELP1b has a longer ELP chain. The position of the ELP block in the fusion was also changed to obtain the new fusion mCherry-ELP0c and ELP1c-mCherry by switching N/C termini of the ELP0a-mCherry and mCherry-ELP1 fusions, respectively.
Plasmid construction. The gene encoding for mCherry was prepared for insertion into the bacterial T7 promoter expression vector pET28a (Novagen EMD Chemicals, Inc. CA) by partial digestion with BamHI and HindIII and ligated to form a new plasmid pET28a/mCherry. The NheI/SpeI fragment of different ELP blocks was then designed and inserted at different positions in the corresponding sites of pET28a/mCherry, resulting
ELP
gene-based
8
different
plasmids,
pET28a/ELP0-mCherry
and
pET28a/mCherry-ELP1 for the production of the 8 different ELP0-based and ELP1based fusions, respectively. All of the recombinant expression plasmids were isolated from E. coli NEB5α cells (NEB, MA) with selection from Kanamycin resistance (50 µg/mL), and the correct constructs were verified by DNA sequencing. The full amino acid and DNA sequences of the fusion proteins are included in Figures S1 – S8.
Protein expression. All expression vectors were then transformed into E. coli tuner cells (BL21-DE3; NEB, MA). Authenticity of the clones was confirmed by DNA sequencing. A total of 5 mL of overnight cultures were grown in LB medium with 50 µg/mL kanamycin at 37 °C in a rotary shaker. These starter cultures were used for inoculation of 1 L of LB cultures with 50 µg/mL kanamycin at a ratio of 1/100 of starter to culture
volume. The expression was further performed at 20 °C for 48 h without induction untill bright pink proteins were observed, and the bacteria were harvested by centrifugation (10,000 g for 20 min at 4 °C). Pellets were stored at -80 °C.
Protein purification. The cell pellets were thawed and resuspended in lysis buffer (MENT buffer: 100 mM NaCl, 10 mM Tris, 1 mM EDTA, 5 mM MgCl2, pH 8.0), incubated with 1 mg/mL lysozyme at 4 oC for 30 min, and sonicated. The lysate was clarified, then thermally cycled between 37 °C and 4 °C three times, and further purified by FPLC under native conditions to preserve the chromophore bond of mCherry. Elution fractions containing purified, bright pink proteins were dialyzed into 20 mM Tris-Cl buffer, pH 8. The purity of the protein was confirmed by denaturing gel electrophoresis (SDS-PAGE) for both mCherry-ELP versions with a yield of ~ 50 mg/L.
MGWGSASGLVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGV PGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGV GVPGVGVPGVGETTSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEI KQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRH STGGMDELYK Figure S1. Conceptual amino acid sequence of ELP0-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASGLVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTSMVSKGEEDNMAIIKE FMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPS DGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQL PGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK Figure S2. Conceptual amino acid sequence of ELP0a-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASGLVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVP GVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVG VPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTSMTG GQQMGRGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLK VTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTV TQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLK LKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKTS Figure S3. Conceptual amino acid sequence of ELP0b-mCherry. The region corresponding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSASMVSKGEEDNMAIIKEFMRFKVH MEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPA DIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQ KKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVN IKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSGLVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGV PGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGVPGVGVPG VGIPGVGVPGVGVPGVGVPGVGVPGVGIPGVGVPGVGVPGVGETTS Figure S4. Conceptual amino acid sequence of mCherry-ELP0c. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVSKGEEDNMAIIKEFMRFKVHME GSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPCDGPVMQK KTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNI KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSVPGVGVPGGGVPGAGVPGVGVPGV GVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGG GVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGG GVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTTS Figure S5. Conceptual amino acid sequence of mCherry-ELP1. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGWGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVT QDSSLQDGEFIYKVKLRGTNFPCDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGM DELYKTSVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPG VGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPG AGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPG VGVPGVGVPGGGVPGAGVPGGGVPGVGTSS Figure S6. Conceptual amino acid sequence of mCherry-ELP1a. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVSKGEEDNMAIIKEFMRFKVHME GSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADI PDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPCDGPVMQK KTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNI KLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSVPGVGVPGGGVPGAGVPGVGVPGV GVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGG GVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGG GVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSVP GVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVP GAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVP GVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVP GGGVPGAGVPGGGVPGVGTSVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAG VPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVG VPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGVPGGG VPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSS Figure S7. Conceptual amino acid sequence of mCherry-ELP1b. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined. MGSSHHHHHHSSGLVPRGSHMASVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGV PGAGVPGGGVPGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGV PGVGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGVPGVGV PGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPGVGTSMTGGQQMGRGSMV SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG EFIYKVKLRGTNFPCDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDA EVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTSS Figure S8. Conceptual amino acid sequence of ELP1c-mCherry. The region corresonding to mCherry is indicated by the bold and italic, and the ELP regions are underlined.
Figure S9.Transmittance as a function of temperature in 1 mg/mL solution indicates the thermoresponsive transition of ELP-mCherry fusions (a) mCherry-ELP0, (b) mCherryELP0a, (c) mCherry-ELP0b, (d) ELP0c-mCherry, (e) mCherry-ELP1, (f) mCherryELP1a, (g) mCherry-ELP1b, and (h) ELP1c-mCherry, where high transmittance (below the transition temperature, Tt) indicates individual molecules dispersed in solution, and low transmittance (above Tt) indicates the formation of macrophase separated structures.
Figure S10. Results of turbidity measurements on fusion proteins samples at 50 wt% in water. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherryELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherry-ELP1b, (h) ELP1cmCherry.
Figure S11. Results of bireinfringence measurements on fusion proteins samples at 50 wt% in water. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherry-ELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherry-ELP1b, (h) ELP1c-mCherry.
Figure S12. SAXS as a function of temperature shows different phase behaviors of mCherry-ELP fusion proteins at 50 wt%. From bottom to top, the curves were taken at 10 o C, 15 oC, 20 oC, 25 oC, 30 oC, 35 oC, and 40 oC . (a) ELP0-mCherry, (b) ELP0amCherry, (c) ELP0b-mCherry, (d) mCherry-ELP0c, (e) mCherry-ELP1, (f) mCherryELP1a, (g) mCherryELP1b, and (h) mCherry ELP1c.
Figure S13. Fusion protein absorbance spectra before dehydration, in film, and after rehydration, demonstrating the preservation of mCherry fluorescence for different fusion designs. (a) ELP0-mCherry, (b) ELP0a-mCherry, (c) ELP0b-mCherry, (d) mCherryELP0c, (e) mCherry-ELP1, (f) mCherry-ELP1a, (g) mCherryELP1b, and (h) mCherry ELP1c.
Table S1. Summary of full width half maxima and domain spacings calculated based on fits to peaks in SAXS data for all fusion proteins at 10 oC, 20 oC and 40 oC Fusion ELP0-mCherry
ELP0a-mCherry
ELP0b-mCherry
mCherry-ELP0c
mCherry-ELP1
mCherry-ELP1a
mCherry-ELP1b
ELP1c-mCherry
Temperature (oC) 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40
Peak 1 d (nm) FWHM (nm-1) 28.11 ± 0.08 0.0301 ± 0.0006 28.14 ± 0.09 0.0279 ± 0.0006 24.78 ± 0.04 0.0203 ± 0.0004 24.54 ± 0.07 0.0156 ± 0.0006 25.26 ± 0.08 0.0223 ± 0.0007 18.08 ± 0.08 0.125 ± 0.001 18.16 ± 0.08 0.111 ± 0.001 18.83 ± 0.02 0.0199 ± 0.0003 23.58 ± 0.01 0.0114 ± 0.0001 23.19 ± 0.02 0.0150 ± 0.0002 17.2 ± 0.3 0.327 ± 0.005 16.8 ± 0.3 0.353 ± 0.005 16.5 ± 0.3 0.314 ± 0.006 40.2 ± 0.5 0.113 ± 0.002 40.5 ± 0.8 0.121 ± 0.003 40.3 ± 0.9 0.126 ± 0.003 16.7 ± 0.2 0.220 ± 0.004 16.6 ± 0.2 0.222 ± 0.004 16.8 ± 0.2 0.214 ± 0.004
Table S2. Molecular weights of proteins as measured by MALDI-TOF compared to calculated protein molecular weight based on sequence Fusion ELP0-mCherry ELP0a-mCherry ELP0b-mCherry mCherry-ELP0c mCherry-ELP1 mCherry-ELP1a mCherry-ELP1b ELP1c-mCherry
Molecular Weight Calculate by Sequence (kDa) 69.4 71.8 47.1 71.8 46.3 43.9 73.7 46.3
Molecular Weight Measured by MALDI-TOF (kDa) 69.0 72.1 49.3 72.0 47.1 40.9 72.2 46.7
