University of Groningen

Thermotropic liquid crystals from engineered polypeptidesPesce, Diego

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Chapter 2

ELP Based Thermotropic Liquid Crystals This chapter has been published as part of: Kai Liu, Dong Chen, Alessio Marcozzi, Lifei Zheng, Juanjuan Su, Diego Pesce, Wojciech Zajaczkowski, Anke Kolbe, Wojciech Pisula, Klaus Müllen, Noel A. Clark and Andreas Herrmann Proceedings of the National Academy of Sciences, 111, 18596–18600 (2014).

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Abstract Here, we demonstrate a simple generic method for the production of water-free thermotropic liquid crystals (TLCs) based on elastin-like polypeptides (ELPs). ELPs were genetically engineered, to carry 72 negative charges, by introducing glutamic acid within the elastin pentapeptide motif. Anhydrous TLCs were obtained via electrostatic complex formation of negatively charged ELPs (acting as the rigid part) with surfactants containing flexible alkyl tails, followed by dehydration. The liquid crystalline structures formed were analyzed by different techniques such as DSC, POM, SAXS, WAXS and TEM. The ELP-surfactant complex exhibits a transition from isotropic to smectic phase, where single surfactant layers separate the polypeptide lamellae. A second transition to an undulated phase was also observed. This, and low transition temperatures to other phases enable the study of TLC phase behavior without thermal degradation of the biomolecular components.

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2.1 Introduction As mentioned in Chapter 1, liquid crystals (LCs) play an important role in biology because their essential characteristic - the combination of order and mobility - is a basic requirement for self-organization and structure formation in living systems1-3. Generally, LC phases formed by many biomolecules, including nucleic acids4,5, proteins6,7, and viruses8,9, are classified as lyotropic. The general term is applied to LC structures formed in water and stabilized by the distinctly biological theme of amphiphilic partitioning of hydrophilic and hydrophobic molecular components into separate domains. However, the principal thrust and achievement of the study of protein LCs has been in the science and application of thermotropic materials, structures and phases in which molecules that are only weakly amphiphilic exhibit LC ordering by virtue of their steric molecular shape, flexibility, and/or weak inter-molecular interactions (e.g., van der Waals and dipolar forces10). These characteristics enable thermotropic LCs (TLCs) to adopt a wide variety of exotic phases and to exhibit dramatic and useful responses to external forces, including, for example, the electro-optic effects that have led to LC displays and the portable computing revolution. This general distinction between lyotropic and thermotropic LCs suggests that there may be interesting possibilities in the development of biomolecular or bio-inspired LC systems in which the importance of amphiphilicity is reduced and the LC phases obtained are more thermotropic in nature. Such biological TLC materials are very appealing for several reasons. Most biomacromolecules were extensively characterized in aqueous environments but in TLC phases their solvent-free properties and functions could be investigated in a state where no or only traces of water are present. Water exhibits a high dielectric constant and has the ability to form hydrogen bonds, greatly influencing the structure and functions of biomacromolecules or compromising electronic properties like charge transport11-13. Considering the many technologies that are incompatible with aqueous systems (e.g. high- and low-temperature applications), investigation of TLCs from biomacromolecules in a water-free environment would expand their usefulness outside of the set of conditions dictated by biology. Indeed, as mentioned in chapter 1, some anhydrous TLC systems containing glycolipids14-17, ferritin18, and polylysine have been reported19-21. However, a general approach to fabricate TLCs based on polypeptides remains elusive. Elastin is one of the most important classes of naturally occurring structural

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proteins22-27. It is commonly found in the ligaments, arteries, skin and lung tissues of mammals, where it exhibits elastic properties and is capable of deforming to store substantial amounts of mechanical energy28-31. In the last few decades, several different variants of elastin have been genetically engineered and used for various applications like tissue engineering32-35, biomolecular recognition36, biolubrication37, and drug delivery38-42. Up to now, ELPs are usually investigated in aqueous solutions or in the cross-linked hydrogel state when they are swollen with water. ELP fluids (liquid crystals and isotropic liquids) in the absence of solvent have not so far been achieved, though these types of ELP soft materials are very appealing for several reasons. Solvent-free ELP fluids may have negligible volatility, and may exhibit high density ELP packing. They are attractive candidates for use as injectable biomaterials, potentially having the ability to mediate cellular adhesion and proliferation if equipped with the appropriate peptide signals43,44. Moreover, they could serve as a compatible matrix for other organic or inorganic components to enhance their mechanical properties. Furthermore, the development of solvent-free ELP liquid crystals introduces ordering and mobility in the fluid, which may lead to novel ensemble properties, that are significantly different from those of water-based ELP systems or traditional organic LCs. In the absence of water, solid ELP does not transition into a fluid state upon heating, due to their make-up of persistent structures, which exceed the range of their intermolecular forces45. Consequently, solvent-free ELPs only undergo thermal degradation at elevated temperatures. Therefore, the development of a general strategy for fabricating solvent-free ELP fluids (liquid crystals and isotropic liquids) is an attractive goal. Genetic engineering as a recombinant protein synthesis technology is a powerful tool for the synthesis of ELPs with predictable properties and biofunctionality. Engineered ELPs allow fabrication of desired sequences with high molecular weights combined with monodispersity, well-defined structures and chain lengths that are impossible to achieve by chemical synthesis46-48. In this chapter, we demonstrate the creation of genetically engineered ELPs based on the common pentameric repeat sequence (VPGXG)n found in tropoelastin. By taking advantage of the flexibility of the amino acid composition at the fourth position of that peptide motif, we generate a negatively charged ELP carrying 72 negative charges by introducing glutamic acid residues. Inspired by the molecular design principles of TLC materials49, which combine rigid or semi-rigid anisometric units with flexible alkyl chains, we

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successfully realized lamellar-structured TLCs from genetically engineered ELP. A general, simple, and effective strategy was developed whereby negatively charged biomacromolecules act as rigid part, and cationic surfactants comprise the flexible units.

2.2 Results and Discussion

2.2.1 Fabrication of the E72 Genetic engineered negatively charged ELPs were fabricated by substituting the amino acid X in the repetitive sequence (VPGXG) with glutamic acid (Glu). Monomer units of the ELP gene encoded ten pentapeptide repeats (Val-Pro-Gly-Glu-Gly) (VPGEG) (Figure 2a) and were multimerized using recursive directional ligation, as described by Chilkoti and co-workers50 to obtain ELPs with 72 negative charges (hereafter E72).

The ELP E72 was produced in large scale in E. coli and purified. Protein yield was around 5 mg per liter of bacterial cell culture. The purity of the product was confirmed by polyacrylamide gel electrophoresis (Figure 1a). ELP E72

FIGURE 1. CHARACTERIZATION OF ELP E72. a) SDS-PAGE of ELP E72, which is separated on a 12% SDS-PAGE gel and stained with 0.3M copper (II); b) MALDI-TOF mass spectrum of ELP E72.

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exhibited reduced electrophoretic mobility compared to globular proteins, a finding widely observed with ELPs50. Mass spectra yielded a single peak for E72 (Figure 1b) and the determined mass was in excellent agreement with the calculated mass (E72 = 36,512 KDa) based on the amino acid sequence.

2.2.2 Preparation and characterization of the E72-DDAB complex After successful design and expression of E72, our goal was to investigate the formation of water-free TLCs involving the polypeptide and the cationic surfactant. E72 (Figure 2a) was complexed with the cationic surfactant didodecyldimethylammonium bromide (DDAB) (Figure 2b). The ELP-DDAB complex (E72-DDAB) was obtained by direct precipitation at room temperature by mixing two aqueous solutions of E72 and DDAB. The anhydrous E72-DDAB complex was obtained after centrifugation and

Figure 2. REPRESENTATION OF ELP E72 AND DDAB SURFACTANT. a) Schematic representation of ELP E72 with the corresponding amino acid sequence of the pentameric motif (in red the glutamic acid residue that was exchanged from a valine); b) didodecyldimethylammonium bromide (DDAB) surfactant and its chemical structure.

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lyophylization of the precipitate. The composition of the ELP-DDAB complex (the [ELP]:[DDAB] ratio) was determined to be 1:77. This measurement suggests that one surfactant molecule electrostatically interacts with one glutamic acid side chain. Thermogravimetric analysis (TGA) of E72-DDAB complex gave a water content of less than 2%, confirming that only traces of water were present in the bulk material and the thermal degradation starts at around 200 °C (Figure 3a).

As shown in the polarized optical microscopy (POM) image the complex exhibited birefringent focal-conic textures (Figure 4a) that is indicative for a smectic phase. Upon heating above 30 °C, the solid material became a viscoelastic LC with strong birefringence (Figure 4b). Above 80 °C, the ELP-DDAB complex transformed into an isotropic liquid but was still viscous due to the ELP’s inherent elastic and mechanical properties47. POM control experiments with pure DDAB51 showed only a long range ordered polycrystalline structure below 60 °C (Figure 4c) and distinct birefringence (Figure 4d) that persisted across the temperature range investigated51 up to 90 °C. The mesophases could be reversibly induced and, when the E72-DDAB complex was cooled from the isotropic phase again, exhibited the same birefringent focal-conic textures.

FIGURE 3. CALORIMETRIC ANALYSIS OF E72-DDAB COMPLEX. a) TGA analysis of E72-DDAB complexes. A water content of less than 2% was measured, confirming only traces of water present in the bulk material and the thermal degradation starts at around 200 °C; b) DSC traces of ELP-DDAB complexes with different transition temperatures. Comparison DSC graphs between E72-DDAB (black line) and DDAB alone (red line) shows that the complex has lower phase transition temperatures.

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These experiments suggested that engineered positively charged ELPs form TLCs in the absence of any solvent although they did not exhibit a typical α-helical or β-sheet secondary structure, as was demonstrated previously42. The long unfolded and charged peptide chains form smectic TLC phase in the absence of water or any other organic solvent, even though the molecules lack sufficient anisotropy according to Onsager’s criterion for orientational ordering52. E72 could be transferred from the TLC to the aqueous phase by treatment with saturated NaCl solution. This result shows that ELP formed lamellar ordered solvent-free TLCs by employing electrostatic interactions between the unfolded peptide chains and the surfactant (Figure 5a). This organization behavior is significantly different from lyotropic LC systems, where only ds DNA or RNA, and α-helical or β-sheet proteins organize into ordered structures in solution1.

FIGURE 4. POLARIZED OPTICAL MICRISCOPE IMAGES. a) POM images of the mesophase cooled from isotropic of the E72-DDAB complex at 35 °C showing well-defined focal-conic textures of smectic layers; b) POM image showing the strong birefringence of viscous liquid crystalline E72-DDAB complex after heating above 30 °C; c) POM image of pristine recrystallized DDAB after cooling to 55 °C showing needle like textures; d) POM image showing the persistent birefringence of pristine DDAB after heating to 100 °C.

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2.2.3 Differential Scanning Calorimetry In agreement with the POM analysis, differential scanning calorimetry (DSC) scans showed two main phase transitions for the peptide complex at about 30 and 75 °C, corresponding to crystalline–smectic and smectic–isotropic transitions, respectively (Figure 3b). Significant differences of the LC behavior were detected for ELP-DDAB complexes and for the pristine DDAB surfactant (used as a control). DDAB melts at 61 °C, which is 30 °C higher than the ELP-DDAB complexes. At 75 °C, the ELP-DDAB complexes exhibited an isotropic liquid phase. However, DDAB alone has a second endothermic peak at 77 °C but, as discussed above, still gives distinct birefringence (Figure 4d) across the temperature range investigated51. The decrease of phase transition temperatures of ELP-DDAB complexes compared to the pristine surfactant indicates that intermolecular interactions between DDAB become weaker due to the presence of unfolded polypeptides in the LC.

2.2.4 SAXS and WAXS To obtain deeper insights into the polypeptide based TLC structures, small-

FIGURE 5. PROPOSED STRUCTURES OF THE E72-DDAB COMPLEX. Sketches of the proposed structures of thermotropic liquid crystals (TLCs) formed by ELP E72 complexed with surfactants DDAB (E72-DDAB) showing a lamellar phase a) the bilayer smectic phase and b) the modulated smectic (Smmod) phase that is observed at lower temperature and the corresponding phase transition temperatures. The lamellar bilayer structures are made of alternately one sublayer of the biomacromolecule and one interdigitated sublayer of the surfactants, where the negatively charged glutamate residues of E72 electrostatically interact with the cationic head groups of the surfactants.

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angle and wide-angle X-ray scattering (SAXS and WAXS) profiles of E72-DDAB (Figure 6 and 7) were measured. The WAXS profile of the complex after formation, as precipitated powder, at room temperature (25 °C) showed polycrystalline nature (Figure 7c). After heating the complex and cooling down, at 70 °C SAXS (Figure 6a and 7a) results showed a well-defined maximum of the first order diffraction at 0.260 Å-1, as well as the corresponding second and third order diffractions at 0.521 and 0.779 Å-1, respectively. The correspondent layer spacing is of d = 24.1 Å and it is in agreement with the thickness of the ELP layer (∼ 10 Å) and the DDAB

monolayer without fully extended tails (∼14 Å)53. These results confirm a bilayer structure of the mesophase, with alternatingly an ELP sublayer (d∼10 Å) and an interdigitated DDAB sublayer (d∼14.4 Å) (Figure 5a and 6a inset). WAXS results (Figure 6b, d) exhibited a typical broad peak of the lipid at about 4.5 Å, confirming the intralayer packing of the DDAB lipids.

FIGURE 6. SAXS AND WAXS PROFILES OF THE E72-DDAB COMPLEX. SAXS (a, c) and WAXS (b, d) profiles of the biomolecule-surfactant complexes cooled from isotropic to the smectic mesophases. The corresponding molecular organizations of the different smectic structures (side view) are given in the insets. (a, b) E72-DDAB at T=70°C; (c, d) E72-DDAB at T=35°C. The multiple peaks (q = 0.196, 0.205, 0.227, 0.249 Å-1) can be indexed as (s=1, m=0), (1, 1), (1, 2), (1, 3) where s is along the layer normal and m is in the layer plane, indicating an undulated smectic structure of two-dimensional ordering. In c, the 2nd order scattering (q~0.402 Å-1) of E72-DDAB is overlapped with that of kapton (which is used for sample loading and sealing).

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X-ray control experiments with pure DDAB, as carried out for POM results51, showed only a long range ordered polycrystalline structure below 60 °C (Figure 8). When cooling below T=55 °C, the layer spacing increases from d = 24.4 Å to d = 31.4 Å (Figure 6c and 7b) and a set of subsidiary satellite peaks (q = 0.205, 0.227, 0.249 Å-1) appears around the smectic layer reflection peak, which are

FIGURE 7. SAXS AND WAXS PATTERNS OF THE E72-DDAB COMPLEX. SAXS pattern of the unoriented E72-DDAB complex at 70 °C a) and 35 °C b) upon cooling down. In pattern a, the diffraction rings at q1, q2, and q3 are the first, second and third orders of the 24.15 Å layer spacing, whereas the broad ring between q1 and q2 corresponds to the kapton diffraction. In pattern b, besides the main diffractions (q1, q2, q3, q4) of the lamellar phase, additional scattering rings between q1 and q2 appear (black arrow, 0.227 Å-1 and 0.249 Å-1),indicating the undulated lamellar structure at low temperatures. It is necessary to point out that the second scattering of the mesophase is overlapped with the kapton diffraction at 35 °C; c) WAXS profiles of the unoriented E72-DDAB complex at 25 °C indicating polycrystalline properties.

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indexed as (1, 1), (1, 2) and (1, 3) of a two-dimensional (2D) rectangular lattice. These data suggest an additional periodicity perpendicular to the layer normal, i.e. an in-plane layer undulation that, with the layering forms a type of LC columnar phase.

Figure 7a and 7b show the SAXS patterns of the E72-DDAB complex at 70 °C and 35 °C upon cooling. In pattern a, the diffractions marked with arrows at q1, q2, and q3 are the first, second and third orders of the 24.15 Å layer spacing. The intensity anisotropy indicates partial orientation of the sample with a layer normal in the y-z plane. The broad ring between q1 and q2

FIGURE 8. SAXS PROFILE OF DDAB. SAXS profiles of the recrystallized DDAB alone upon cooling down to 55 °C (a, b) and 35 °C (c, d). These results indicate that DDAB below 60 °C was already become a polycrystalline solid.

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is from the kapton tape, which is used for sample loading and sealing. In pattern b, besides the q10, q20 and q30 diffraction from the layering, the strong q1,3 peak shown in figure 6c also appears, indicating the 2D columnar lattice formed by the layering and layer modulation at low temperatures. In this case, the columnar domain has the layer/modulation columns running parallel to the z direction, i.e. they are also aligned by, and parallel to, the surface. The layering, qs, and modulation, qm, wavevectors of the 2D columnar lattice are in the y-z plane, as is q1,3 = qs +/- 3qm. Moreover, the small peak at around 50-55 °C observed in the DSC scan (Figure 3b) confirms a mesophase transition between a simple lamellar architecture (55–75 °C) and the undulated smectic structure (31-55 °C). Interestingly, these results together, indicate that the complex undergoes an Isotropic-Smectic-Modulated Smectic (Iso-Sm-Smmod) phase sequence (Figure 5). Figure 5 represents a schematic model of the Iso-Sm-Smmod phase transition sequence.

2.2.5 FF-TEM In accordance with the above analysis, visualization of the undulated lamellar structure was realized by Freeze-Fracture TEM (FF-TEM). As shown in Figure 9a, the smectic fracture surfaces exhibit the long ordered lamellar

FIGURE 9. FF-TEM OF E72-DDAB COMPLEX. FF-TEM images of the mesophases formed by the E72-DDAB complex. The microstructures of the Smmod mesophases observed in the E72-DDAB complexes showing a 2D periodic lattice: the smectic layers parallel to the image plane and the in-plane layer undulation (a, top view; b, side view).

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structures and well-defined layer steps were detected. Smectic layer surfaces are generally smooth but have occasional layer steps that are distinct and can be identified unambiguously. In addition to the lamellar structure, periodic in-plane layer undulation was also directly observed, (Figure 9b), and the multiple-step “cliffs” (Figure 9b) show the ripples that propagate through many layers without changing the smectic structure. Periodicities of 17 nm, close to the x–ray in-plane modulation wavelength 12 nm, and 50 nm are evident in the FFTEM images (Figure 9b). This kind of layer undulation has so far been observed in the ripple phase of phospholipids54 and in the B1 and B7 phases of bent-core liquid crystals55-57.

2.3 Conclusion In this work we demonstrated the realization of solvent-free thermotropic liquid crystals from genetically engineered ELPs. We designed an elastin variant with a negative charge in every pentapeptide repeat, resulting in a polyelectrolyte with 72 charges (E72) that is perfectly defined regarding the number and distribution of charges, amino acid composition, stereochemistry and dispersity, which is almost impossible to accomplish by conventional polymerization techniques. E72 was complexed with the cationic surfactant didodecyldimethylammonium bromide (DDAB) and successively dehydrated. This simple fabrication procedure forms a smectic thermotropic LC material thanks to charge-charge interactions. The electrostatic complex was stable, uniform and at the same time self-assembled into a multilayer architecture. The anhydrous smectic phases that result exhibit biomacromolecular sublayers intercalated between aliphatic hydrocarbon sublayers at, or near, room temperature. The material showed two main phase transitions at about 30 °C and 75 °C, corresponding to crystalline–smectic and smectic–isotropic transitions. An additional mesophase transition was detected between a simple lamellar architecture (55–75 °C) and the undulated smectic structure (31-55 °C) with DSC and X-ray analysis and visualized under TEM. This is the first realization of ELP fluids (liquid crystals and isotropic liquids) in the absence of water. Moreover, it is worth to note that they assemble into periodic lamellar structures in spite of the fact that these molecules lack sufficient rigidity. Genetic engineering is a powerful tool to fabricate de novo designed polypeptide building blocks that are constituent components of TLCs. In the

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future, we will study the properties of the biological components within this novel class of TLCs providing a hydrophobic environment and lacking high water content and investigate if it allows integration of other functional proteins, which have no LC properties itself.

2.4 Experimental Section

2.4.1 Materials E. coli XL1-Blue competent cells were purchased from Stratagene (La Jolla, CA). The pUC19 cloning vector, restriction endonucleases, T4 DNA ligase (LC), Fast APTM thermosensitive alkaline phosphatase (Fast AP), and GeneJETTM Plasmid Miniprep kit were purchased from Fermentas (St. Leon-Rot, Germany). Digested DNA fragments were purified using QIAquick® spin miniprep kits from QIAGEN, Inc. (Valencia, CA). The pET-25b(+) vector and E. coli BLR(DE3) competent cells were purchased from Novagen Inc. (Milwaukee, WI). Oligonucleotides were synthesized by biomers.net (Ulm, Germany). BactoTM tryptone and BBLTM yeast extract were purchased from Becton, Dickinson and Co. (Sparks, MD). Potassium phosphate monobasic, potassium phosphate dibasic, sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride, and glycerol were purchased from Merck KGaA (Darmstadt, Germany). Ampicillin and imidazole were purchased from Roth (Karlsruhe, Germany). Isopropyl ß-thiogalactopyranoside (IPTG) was purchased from Duchefa (Harlem, Netherlands). 3,5 dimethoxy-4-hydroxycinnamic acid and internal standards bovine serum albumin and trypsinogen were purchased from LaserBio Labs (Sophia-Antipolis, France). Surfactant didodecyldimethylammonium bromide (DDAB) was purchased from ABCR (Germany). All solvents and reagents were used without further purification. During all experiments, ultrapure water (18.2 MΩ) purified by MilliQ-Millipore system (Millipore, Germany) was used.

2.4.2 Methods Cloning/Gene oligomerization The building block of the anionic ELP gene (E9) (Figure 10a) was ordered

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from Entelechon (Regensburg, Germany) and was delivered in the pEN vector. Gene sequence and the respective amino acid sequence of the monomer is shown in figure 10. As the recognition sites of the restriction enzymes PflMI and BglI had to be preserved, one valine residue per ten pentapeptide repeats was incorporated instead of a glutamic acid residue.

All cloning steps were performed according to standard molecular biology methods. The ELP gene was excised from the pEN vector by digestion with EcoRI and HinDIII and run on a 1% agarose gel in TAE buffer (per 1L, 108 g Tris base, 57.1 mL glacial acetic acid, 0.05 M EDTA, pH 8.0). The band containing the ELP gene was excised from the gel and purified using a spin

FIGURE 10. ELP E72 OLIGOMERIZATION. a) Gene and corresponding polypeptide sequence of ELP E9. Recognition sites for the restriction enzymes EcoRI, PflMI, BglI, and HindIII are underlined; b) Gel electrophoresis of ELP genes. PUC vectors containing the ELP genes were digested with EcoRI and HinDIII and separated on a 1% agarose gel. DNA bands were visualized by ethidium bromide staining. Digestion produced a vector fragment of 2,635 bp and an ELP gene fragment. n = number of monomers with 10 pentapeptide repeats per monomer; E = negatively charged; M: size standard.

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column purification kit. PUC19 was digested with EcoRI and HinDIII and dephosphorylated. The vector was purified by agarose gel extraction following gel electrophoresis. The linearized pUC vector and the ELP-encoding gene were ligated and transformed into chemically competent XL1-Blue cells according to the manufacturer’s protocol. Cells were plated and colonies were picked and grown in LB medium supplemented with 100 µg/mL carbenicillin overnight, and plasmids were isolated using the GeneJET Plasmid Miniprep kit. Positive clones were verified by plasmid digestion with EcoRI and HinDIII and subsequent gel electrophoresis (Figure 10b). Gene oligomerization was performed as described by Chilkoti and co-workers50 (Figure 10). The DNA sequence of putative inserts was further verified by DNA sequencing (SequenceXS, Leiden, The Netherlands). Expression vector construction The expression vector pET 25b(+) was modified by cassette mutagenesis, for incorporation of a unique SfiI recognition site and an affinity tag consisting of six histidine residues at the C-terminus (Figure 11), as described before42.

The modified pET 25b(+) vector (henceforward called pET-SfiI-H6) was digested with SfiI, dephosphorylated and purified using a microcentrifuge spin column kit. The ELP gene was excised from pUC19 vector by digestion with PflMI and BglI and purified by agarose gel extraction following gel electrophoresis. The linearized vector and ELP gene were ligated, transformed into XL1-Blue cells, and screened as described above.

FIGURE 11. SEQUENCE INSERTED INTO pET-25b(+). The sequence inserted into pET-25b(+) between recognition sites NdeI and EcoRI. The modified pET-SfiI-H6 vector contains a unique SfiI recognition site to insert ELP genes into the vector, and sequence encoding for a hexa-histidine (H6) tag at the C-terminus of the expressed protein for affinity purification.

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Protein expression and purification E. coli BLR (DE3) cells were transformed with the pET-SfiI-H6 expression vectors containing the respective ELP gene. For protein production, Terrific Broth medium (for 1 L, 12 g tryptone and 24 g yeast extract) enriched with phosphate buffer (for 1 L, 2.31 g potassium phosphate monobasic and 12.54 g potassium phosphate dibasic) and glycerol (4 mL per 1 L TB) and supplemented with 100 µg/mL ampicillin, was inoculated with an overnight starter culture to an initial optical density at 600 nm (OD600) of 0.1 and incubated at 37 °C with orbital agitation at 250 rpm until OD600 reached 0.7. Protein production was induced by a temperature shift to 30 °C. Cultures were then continued for additional 16 h post-induction. Cells were subsequently harvested by centrifugation (7,000 x g, 20 min, 4 ºC), resuspended in lysis buffer (50 mм sodium phosphate buffer, pH 8.0, 300 mм NaCl, 20 mм imidazole) to an OD600 of 100 and disrupted with a constant cell disrupter (Constant Systems Ltd., Northands, UK). Cell debris was removed by centrifugation (40,000 x g, 90 min, 4 ºC). Proteins were purified from the supernatant under native conditions by Ni-sepharose chromatography. Product-containing fractions were pooled and dialyzed against ultrapure water and then purified by anion exchange chromatography using a Q HP column. Protein-containing fractions were dialyzed extensively against ultrapure water. Purified proteins were frozen in liquid nitrogen, lyophilized and stored at -20 ºC until further use. Protein Characterization The concentrations of the purified ELPs were determined by measuring absorbance at 280 nm and fluorescence spectra were both measured using a SpectraMax M2 instrument (Molecular Devices, Sunnyvale, CA). Protein purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel stained with 0.3 M copper (II) chloride. Photographs of the gel was taken with a LAS-3000 Image Reader (Fuji Photo Film (Europe) GmbH, Dusseldorf, Germany). Mass Spectrometry Mass spectrometric analysis was performed using a 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems, Foster City, CA, USA) in the linear positive mode. The protein samples were mixed 1:1 v/v with a recrystallized α-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 50% ACN and 0.1% TFA). Trypsinogen (MW = 23,980 KDa), enolase (MW = 46,672 KDa) and bovine

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serum albumin (MW = 66,431 KDa) were used as calibration standards. Mass spectra were analyzed and calibrated internally with the Data Explorer software, version 4.9. Preparation of E72-DDAB complexes 36 nmol (1.3 mg) of dry E72 were dissolved in 100 µL NaCl (100 mM) solution. At the same time, ~3 mole equivalents of cationic DDAB relative to glutamate residues within the ELP were dissolved in an equal volume of ultrapure water. After mixing of the two solutions a precipitate occurred immediately. The precipitate was collected by centrifugation and was then lyophilized before further characterization. The composition of the E72-DDAB complex was determined as described below. First the lyophilized sample was weighed by a high precision electronic balance to measure the total weight of the material. Then the E72 concentration was obtained by measuring the UV absorption (280 nm) after dissolving the sample in chloroform. From the E72 concentration and total weight, the [E72]:[DDAB] ratio was calculated. POM POM (polarized optical microscopy) was conducted on a Zeiss Axiophot using a temperature program with a heating/cooling rate of 5 °C/min. The measurements were carried out on samples sandwiched between clean glass microscope slides spaced by a gap of 10 µm. DSC DSC (differential scanning calorimetry) was carried out using a TA Instruments Q1000 in a nitrogen atmosphere and with the same temperature program as employed in the POM experiment (heating/cooling rate of 5 °C/min). SAXS and WAXS Small-angle and wide-angle X-ray scattering (SAXS and WAXS) with heating and cooling systems was performed by employing a conventional X-ray source. For SAXS, a Bruker Nano/microstar machine with radiation wavelength of λ = 1.54 Å was used to obtain small angle scattering profiles, where the sample-to-detector distance was 240 cm. The sample holder is a metal plate with a small hole (diameter ~0.25 cm, thickness ~0.15 cm), where the X-ray beam passes through. The samples are loaded without any

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orientational treatment in the hole and are sealed by kapton tape. The scattering vector q is defined as q = 4π•sinθ/λ with 2θ being the scattering angle. WAXS was carried out by a home-made, rotating-anode–based setup. The goal of such WAXS experiments was to probe halo diffusion of alkyl chains of surfactants. The sample-to-detector distance was 13 cm with X-ray wavelength of 1.54 Å. SAXS and WAXS profiles were obtained by cooling the samples (5 °C/min) from the isotropic phase to a selected temperature of the mesophase. FF-TEM Freeze-fractured transmission electron microscopy (FF-TEM) was prepared according to standard protocols58. It was carried out by sandwiching the samples between 2 mm by 3 mm glass planchettes and cooling from the isotropic melt to a selected temperature in the LC range. The samples were then rapidly quenched to T < -180 °C by immersion in liquid propane, fractured in a vacuum at -140 °C, and then coated with 2 nm of platinum deposited at 45° and then with 25 nm of carbon deposited at 90°. After dissolving the liquid crystal, the Pt–C replicas are placed in the TEM, where the topographic structure of the fracture plane may be observed. TEM was performed on a Philips CM10 transmission electron microscope operating at an accelerating voltage of 100 kV. Images were recorded on a Gatan slow-scan CCD camera.

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