A Domain Swapping Study of Nano-Capsule Proteins. Rongli Fan, Aimee L. Boyle, Vee Vee Cheong, See Liang Ng, Brendan P. Orner*

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1 A Domain Swapping Study of Nano-Capsule Proteins Rongli Fan, Aimee L. Boyle, Vee Vee Cheong, See Liang Ng, Brendan P. Orner* Figure S1. Amino acid sequences of proteins used in this study. Grey shading indicates each of the helices (A-D in order from the N- to C-termini) in the four-helix bundle from the parent protein. A black box indicates the E helix from BFR, and a white box indicates the BC helix from DPS. S1

2 Figure S2. Schematic of gene production and cloning strategy. Figure S3. BFR gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. S2

3 Figure S4. BFR-E gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. Figure S5. BFR+BC gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. S3

4 Figure S6. BFR+BC-E gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. Figure S7. DPS gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. S4

5 Figure S8. DPS-BC gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. Figure S9. DPS+E gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. S5

6 Figure S10. DPS-BC+E gene assembly and amplification: A) The amino acid sequence of the protein. B) The DNA sequence of the forward and reverse oligonucleotides used in gene assembly. C) The DNA sequence of the sense and antisense primers used in the extension PCR to provide LIC termini for cloning. Oligonucleotide Design Short oligonucleotides (synthesized by solid phase phosphoramidite technology by 1 st Base Pte. Ltd., Singapore) were designed based on the amino acid sequences of the native ferritins with codons changed in preference for those that have high usage in E. coli. The oligos were designed so that the 5 strands overlapped by 12 bp, whereas the 3 strands had a 15 bp overlap. The overlap and the C/G content were designed so that the oligos all had similar melting temperatures. S6

7 Figure S11. DNA electrophoretic analysis of the products of gene assembly and amplification. The expected gene sizes for each protein are in Figures S3-S10. S7

8 Figure S12. DNA electrophoretic analysis of the products of extension PCR with LIC primers presented in Figures S3-S10. The band of the appropriate size was excised and purified. S8

9 Figure S13. DNA electrophoretic analysis of the products of single-colony PCR screens for inserted gene after cloning into the expression vector. S9

10 Figure S14. Schematic of protein purification including the alternative on-resin refolding technique. S10

11 Figure S15. Expression and purification of ferritins and ferritin mutants. Lane 1 (from left to right): Protein ladder. Lane 2: Uninduced. Lane 3: Induced. Lane 4: Insoluble fraction. Lane 5: Soluble fraction. Lane 6: Flow through after applying soluble fraction to resin. Lane 7: Flow through from washing the resin. Lane 8: Purified protein after Ek cleavage of the affinity tags and release from the resin. NOTE: The bottom gel for BFR+BC-E is after on-resin refolding. Lane 1:Ladder. Lane 2: Uninduced. Lane 3: Induced. Lane 4: Solubilized lysate. Lane 5: Flow through after applying solubilized lysate to resin. Lane 6: Flow through from washing the resin. Lane 7: Purified protein after Ek cleavage of the affinity tags and release from the resin. The bottom gel for DPS-BC+E is after on-resin refolding. Lane 1: Ladder. Lane 2: Uninduced. Lane 3: Induced. Lane 4: Solubilized lysate. Lane 5: Flow through after applying solubilized lysate to resin. Lane 6: Flow through from washing the resin #1. Lane 7: Flow through from washing the resin #2. Lane 8: Flow through from washing the resin #3. Lane 9: Purified protein after Ek cleavage of the affinity tags and release from the resin. S11

12 Figure S16. Circular dichroism (CD) studies of the ferritins and ferritin mutants. CD spectra of the proteins at different temperatures for BFR (left) and DPS series (right). Spectra were taken for each protein at temperatures from C at 5 C intervals. These spectra were used to determine the loss of helical structure with temperature by following the signal at 222nm and fitting the resulting data (Figure 2) to establish a melting point. (Figure S17) These spectra are the average of at least three replicates melts. S12

13 Figure S17. Melting temperatures from the ferritins and ferritin mutants. These values were obtained by fitting the isotherms in Figure 2. Dark and light grey indicate the BFR and DPS series respectfully. Error bars represent standard error of at least three replicates. S13

14 Figure S18. Folding reversibility of the ferritins and ferritin mutants. Circular Dichroism spectra at 20 C before the melting experiments depicted in Figure S16 (black) and at 20 C after the experiment (red). For the BFR (right) and DPS (left) series. S14

15 Figure S19. Size exclusion chromatography calibration. Top: Chromatogram of calibration proteins. Bottom: Stokes radius (Rst) correlation to Kav as defined in the Experimental Section. Conalbumin was excluded from the Stokes radii correlation due to the fact that its radius is unknown. S15

16 Figure S20. Transmission electron micrographs of ferritin and ferritin mutant negatively stained with uranyl acetate. The scale bar is 50 nm. S16