Temperature-Dependent Morphology of Hybrid Nanoflowers. from Elastin-Like Polypeptides

Transcription

1 Temperature-Dependent Morphology of Hybrid Nanoflowers from Elastin-Like Polypeptides Koushik Ghosh, 1 Eva Rose M. Balog, 1 Prakash Sista, 1 Darrick J. Williams, 1 Daniel Kelly, 2 Jennifer S. Martinez,*,1 and Reginaldo C. Rocha*,1 1 Center for Integrated Nanotechnologies (MPA-CINT), 2 Chemistry Division (C-CDE), Los Alamos National Laboratory, Los Alamos, New Mexico, NM 87545, USA * Authors to whom correspondence should be addressed. jenm@lanl.gov; rcrocha@lanl.gov Table of Contents Methods Mass spectra of ELP-2 Determination of the transition temperature of ELP-2 by DLS Additional SEM images of nanoflowers (wide-field view) Additional SEM images of nanoflowers (ELP concentration) Control SEM images: CaCl 2 and CuSO 4 Control SEM images: ELP XRD data XPS survey scan EDAX data 1/12

2 Methods ELP expression and purification ELP amino acid sequence molecular weight (kda) ELP-1 AG[VPGIG] 25 VPASGKPIPNPLLGLDST[H] ELP-2 (His-free) AG[VPGIG] 25 VPASW 11.3 The ELP gene AG[VPGIG] 25 VP was synthesized by GeneArt and was subcloned using BsshII and NheI restriction enzymes into periplasmic expression vectors using standard molecular biology techniques. For ELP-1, the ELP was subcloned into the vector POE that appends C-terminal SV5 and 6XHis tags. For ELP-2, we modified POE to create the POE-W vector which provides a single Trp residue tag. Expressed proteins were directed to the periplasm using a vector-provided pelb leader signal peptide (not shown) that is removed upon secretion. Successfully cloned constructs were verified by sequencing (MWG Operon; sequences available upon request). The choice of isoleucine as a guest residue (VPGXG) in combination with the length of 25 repeats provided a highly practical design with working concentrations in the range 1-10 mg/ml, which allows conservation of protein material while also allowing rapid phasetransition behavior at laboratory-available temperatures of 4 o C and 37 o C in standard buffer conditions. The linker sequence between (VPGIG) 25 and the His-tag is an SV5 (Simian-Virus 5) epitope sequence tag. The SV5 tag provides an additional means of antibody-based detection or immobilization for this protein construct, a feature that was not utilized in this study but provides a versatile biochemical handle for future experiments. ELP expression and purification protocols were adapted from Hassouneh et al. 1 Plasmids encoding ELPs were transformed into competent BL21(DE3) E. coli (NEB). Transformed cells 1) W. Hassouneh, T. Christensen, and A. Chilkoti, Curr. Protoc. Protein Sci. Chapter 6, unit 6.11 (2010). 2/12

3 were plated on selective solid medium (2XYT + agar + carbenicillin) overnight. Multiple colonies were transferred to ml starter cultures which were then used to inoculate 1 L 2XYT cultures for 24 h at 37 ºC without induction. Cells were harvested by centrifugation and the periplasmic fraction was obtained by cold osmotic shock in 20% sucrose buffer. ELPs were purified by inverse temperature cycling. Protein purity was assessed by SDS-PAGE and ESI-MS. ELP-1 was visualized using the Coomassie-based stain GelCode Blue (Pierce); ELP-2 did not absorb Coomassie and was visualized using 0.3 M CuCl 2 (data not shown). Following purification, ELPs were dialyzed into nanopure H 2 O, lyophilized, and stored at -20 ºC. Dynamic light scattering (DLS) DLS was performed on a temperature-controlled Zetasizer-Nano dynamic light scattering instrument (Malvern) equipped with a He-Ne laser (633 nm). Data were acquired at 4 ºC intervals from 4 ºC to 40 ºC. After equilibration at each temperature, three volume measurements were taken of a single 0.25 mg/ml protein sample in sterile filtered PBS and the average hydrodynamic diameter was plotted (error bars = ± standard deviation). Mass spectrometry Electrospray ionization time-of-flight (ESI-TOF) mass spectra were collected using a SYNAPT G2 instrument (Waters) with the capillary voltage set to 2.8 kv, cone voltage at 10 V, and source temperature at 100 C. Scanning electron microscopy (SEM) SEM analysis was performed with a FEI Quanta 400 FEG-E-SEM environmental microscope (resolution 3-4 nm, high voltage range from 500V-30kV) and a Magellan 400 high-resolution field-emission microscope (resolution 1 nm) after sputter coating with gold. SEM images were collected from a range of voltage spanning from kv. In SEM experiments, a suspension of nanoflowers was filtered, washed with water and dried on a membrane (pore size 0.25 µm). The sample was subsequently transferred to a silicon grid and sputter coated with gold. 3/12

4 X-Ray Diffraction (XRD) XRD measurements were made on a Rigaku Ultima III diffractometer that uses a fine line sealed Cu tube Kα (λ= Ǻ) X-rays. The generator is a D/MAX Ultima series with a maximum power of 3 kw. The samples were placed on the top of a thin-film stage plate that was aligned for maximum sample height and X-ray intensity. Data were collected in continuous scan mode, in a parallel beam slit geometry. Samples were subjected to Cu Kα radiation (λ ~ Å) and scanned from 1 to 60 degrees (2θ) at 0.05 intervals at a rate of 1 /min. A silicon wafer with 200 nm of thermally deposited SiO 2 (22 22 mm) was used as the sample substrate. X-ray photoelectron spectroscopy (XPS) XPS data were collected using a Physical Electronics VersaProbe II system with a base pressure below Pa. A 100 μm, monochromated Al kα X-ray source (1487 ev) rastered over a mm area was used throughout, and photoelectrons were energy-sorted using a hemispherical analyzer. Samples were placed on molybdenum platens for analysis and were at room temperature. XP spectra are reported in terms of binding energy (BE), and instrument calibration was performed in accordance with ASTM procedure. Elemental composition (Figures S8 and S9) was determined using survey scans at a pass energy of 117 ev with peak assignments made using the Ulvac-PHI Handbook of X-ray Photoelectron Spectroscopy. A pass energy of 29 ev was used for high-resolution scans to determine chemical valence state; peak assignments are referenced in the article text. Charge neutralization for insulating samples is accomplished by focusing low-energy ions and electrons at the spot of X-ray impingement. For XPS experiments, one drop of a nanoflower suspension was added onto a silicon grid and dried at 4 C or 37 C. 4/12

5 Mass spectra of ELP-2 (N-AG[VPGIG] 25 VPASW-C) Figure S1: Isotopic distribution and ESI mass spectra of ELP-2 (N-AG[VPGIG] 25 VPASW-C). Determination of the transition temperature of ELP-2 by DLS Figure S2: Dynamic light scattering data for ELP-2 (0.25 mg/ml in PBS). 5/12

6 Additional SEM images of nanoflowers (wide-field view) Figure S3: SEM images with larger, zoom-out views of representative samples of calcium phosphate crystals grown from ELP-1, CaCl 2, and PBS below T t, at 4 o C (A and B) and above T t, at 37 o C (C and D). 6/12

7 Additional SEM images of nanoflowers (ELP concentration) Figure S4: SEM images of representative samples of copper phosphate crystals grown from CuSO 4, PBS, and ELP-1 at the concentration of 0.25 mg/ml (A) and 1.0 mg/ml (B). 7/12

8 Control SEM images: CaCl 2 and CuSO 4 A B Figure S5: SEM images of precipitates from CaCl 2 (A) and CuSO 4 (B) in PBS. 8/12

9 Supplementary Material Control SEM images: ELP A B Figure S6: SEM images of (A) ELP-1 in PBS drop cast onto a silicon grid, and (B) subsequent addition of CuSO4 to the drop cast ELP-1 (in PBS). 9/12

10 XRD data Figure S7: Representative XRD data for nanoflowers grown at 4 o C (i.e., below T t ) from CaCl 2 and ELP-1 in PBS. No significant difference was observed in the data for nanoflowers grown at 37 o C (above T t ). The XRD pattern of the sample matches with Monetite, CaPO 3 (OH) (reference PDF files: and ). 10/12

11 XPS survey scan Figure S8: XPS survey scans of Cu 3 (PO 4 ) 2 grown in PBS, with and without ELP-1. Figure S9: XPS survey scans of Ca 3 (PO 4 ) 2 grown in PBS, with and without ELP-1. 11/12

12 EDAX data A B Figure S10: EDAX data of nanoflowers grown from CaCl 2, ELP-1, and PBS (A) and from CuSO 4 5H 2 O, ELP-1, and PBS (B). [The peak corresponding to Si originates from the sample substrate (silicon grid).] 12/12