Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Jun 13.
Published in final edited form as: Methods Mol Biol. 2017;1586:211–220. doi: 10.1007/978-1-4939-6887-9_13

Protein Folding Using a Vortex Fluidic Device

Joshua Britton 1,2,ψ, Joshua N Smith 3,ψ, Colin L Raston 2,*,†, Gregory A Weiss 1,3,*,†
PMCID: PMC5999337  NIHMSID: NIHMS973082  PMID: 28470607

Abstract

Essentially all biochemistry and most molecular biology experiments require recombinant proteins. However, large, hydrophobic proteins typically aggregate into insoluble and misfolded species, and are directed into inclusion bodies. Current techniques to fold proteins recovered from inclusion bodies rely on denaturation followed by dialysis or rapid dilution. Such approaches can be time consuming, wasteful, and inefficient. Here, we describe rapid protein folding using a vortex fluidic device. This process uses mechanical energy introduced into thin films to rapidly and efficiently fold proteins. With the VFD in continuous flow mode, liters of protein solution can be processed per day with 100-fold reductions in both folding times and buffer volumes.

Keywords: Protein Folding, Vortex Fluidics, Continuous Flow, Misfolded Proteins, Inclusion Bodies, Bacterial Protein Expression

1. Introduction

Protein overexpression provides important tools for a wide range of applications ranging from therapeutics to laboratory reagents (1–3). Bacterial protein overexpression capitalizes upon the low cost, high growth rate, and versatility of Escherichia coli (4, 5). Although many proteins correctly fold during overexpression in E. coli, larger or very hydrophobic proteins often generate misfolded and insoluble aggregates, which are directed into inclusion bodies (6–8). Recovering such aggregated proteins often involves chemical denaturation of the inclusion bodies, then multi-day dialysis to remove the denaturant (9, 10). An alternative method applies high pressure to drive proteins into their folded states (11). Illustrative of the wasteful inefficiency of conventional protein folding, liters of buffer are typically used to refold small quantities (mg) of protein (12, 13). To address this issue, we have developed an approach to harness mechanical energy supplied by a vortex fluid device (VFD) to rapidly fold proteins in vitro under standard conditions.

The VFD, a commercially available benchtop device, can drive a range of chemical transformations in its thin films (14–19). Rapid rotation of a solution in a glass sample tube creates a dynamic thin film with ≈230 μm thickness (16). Within this film, reagents experience micromixing, shear stress, and vibrational effects (19–24). At specific rotational speeds, the sample tube enters a harmonic vibration that generates Faraday or pressure waves in the fluid, which can accelerate protein folding (25, 26).

The method described here is demonstrated with the folding of recombinant hen egg white lysozyme (HEWL) overexpressed in E. coli. The insoluble, misfolded protein results from overinduction, and a correctly folded form can be obtained with short VFD processing times. Though folding HEWL is illustrated here, the approach is generalizable to include other proteins, as demonstrated for cAMP dependent protein kinase A (PKA) and a truncated form of the membrane protein caveolin-1 (27).

2. Materials

2.1 Buffers

All buffers are prepared at 1 L scale with double-distilled H2O (ddH2O) purified to a resistivity of 18 MΩ and filtered through a 0.22 μm filter. Buffers are autoclaved, and then stored at room temperature unless otherwise indicated. All reagents should be at least reagent grade and ACS-certified.

  1. Buffer 1: 50 mM NaH2PO4 and 500 mM NaCl, pH 8.0. To a 1 L Erlenmeyer flask, add 6.00 g of NaH2PO4 and 29.22 g of NaCl. Add approximately 600 mL ddH2O, and dissolve by magnetic stirring. Adjust the pH to 8.0 using 6.0 M NaOH, and transfer the solution to a graduated cylinder before bringing the volume to 1 L with ddH2O (see Note 1).

  2. Buffer 2 (Denaturing buffer): 20 mM Tris-HCl, 10 mM NaCl and 8.0 M urea, pH 8.0. To a 1 L Erlenmeyer flask, add 3.15 g of Tris-HCl, 0.58 g of NaCl, and 480.48 g of urea. Add approximately 300 mL of ddH2O and dissolve the powders into solution using magnetic stirring. Adjust the pH of the solution to 8.0 using 6.0 M NaOH, and transfer to a graduated cylinder before bringing the final volume to 1 L with ddH2O (see Note 2).

  3. Buffer 3 (Elution buffer): 20 mM Tris-HCl, 400 mM NaCl and 8M urea, pH 7.8. To a 1 L Erlenmeyer flask, add 3.15 g of Tris-HCl, 23.38 g NaCl, and 480.48 g of urea. Add approximately 600 mL of ddH2O and dissolve the powders in solution using magnetic stirring. Adjust the pH of the solution to 8.0 using 6.0 M NaOH, and transfer to a graduated cylinder before bringing the final volume to 1 L with ddH2O (see Note 3).

  4. Buffer 4 (Phosphate-buffered saline): 10 mM Na2HPO4, 2.0 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl, pH 7.2. To a 1 L Erlenmeyer flask add 1.42 g of Na2HPO4, 0.27 g of KH2PO4, 0.20 g of KCl, and 8.0 g of NaCl. Add approximately 600 mL of ddH2O and dissolve the powders into solution using magnetic stirring. Adjust the pH of the solution to 7.2 using 6.0 M NaOH, and transfer to a graduated cylinder before bringing the final volume to 1 L with ddH2O.

2.2 VFD Components and Equipment

  1. The VFD, sample tube (20 mm diameter) and delivery jet feeds are purchased from Vortex Fluidic Technologies (www.vortexfluidictechnologies.com).

  2. A laboratory syringe pump with the appropriate tubing (e.g., WPI model AL-1000).

  3. VFD-sample tube caps: B19 Suba-Seals® (Sigma).

  4. Nachi 6005NR open bearings (Nachi-Fujikoshi) (see Note 4).

  5. VFD sample tube lubricant (Dow Corning 976V High Vacuum Grease).

  6. All purpose machine oil (Singer).

2.3 Protein Overexpression, Purification, and Analysis

All reagents for protein overexpression should be molecular biology grade and the appropriate measures taken to maintain sterile working conditions.

  1. Super optimal broth with catabolite repression (SOC): 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4. 20 mM glucose. To a 1 L autoclavable bottle, add 20 g of tryptone, 5 g of yeast extract, 0.584 g of NaCl, 0.186 g of KCl, 0.952 g of MgCl2 and 2.467 g of MgSO4·7H2O. Bring the solution to ≈1 L with ddH2O and swirl to dissolve the solid contents. Autoclave the solution at 121 °C for 20 min. Allow the solution to cool to room temperature, add 3.60 g of glucose, and invert the bottle to mix the contents before use.

  2. Lysogeny Broth (LB): 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl. To a 1 L autoclavable bottle, add 10 g tryptone, 5 g yeast extract, and 10 g NaCl. Bring the final volume to ≈1 L with ddH2O and autoclave at 121 °C for 20 min. Allow the solution to cool to room temperature, and invert the bottle to mix the contents before use.

  3. 40 mg/mL Kanamycin Sulfate: To a 50 mL plastic conical, dissolve 1.60 g of Kanamycin sulfate in approximately 35 mL of ddH2O. Bring the total volume to ≈40 mL with ddH2O. Filter the solution with a 0.22 μm syringe filter, and divide the solution into several 1 mL aliquots in 1.5 mL centrifuge tubes. Store the aliquots at −20 °C.

  4. Sterile petri dish (Fisher).

  5. LB/Kanamycin agar plate: 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 1.5% (w/v) agar. To a 1 L autoclavable bottle, add 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar. Bring the final volume to ≈1 L with ddH2O and autoclave at 121 °C for 20 min. Allow the solution to cool to approximately 55 °C and add 1 mL of the 40 mg/mL kanamycin sulfate from above (giving a final concentration of 40 μg/mL) and swirl to mix. Add 20 mL of this mixed solution to a petri dish (1 L of solution is sufficient for 50 petri dishes). Allow the dishes to cool until the agar has solidified (approximately 1 h), and store at 4 °C.

  6. 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG): In a 50 mL plastic conical, dissolve 5 g of IPTG in 15 mL ddH2O. Transfer to a graduated cylinder, and add ddH2O to a final volume of 21 mL with ddH2O. Filter the solution with a 0.22 μm syringe filter, and divide this into several 1 mL aliquots in 1.5 mL centrifuge tubes. Store the aliquots at ;20 °C.

  7. The gene encoding for HEWL was purchased from Addgene, and was cloned into a pET28c vector (GE Healthcare).

  8. CaCl2 competent E. coli BL21(DE3) cells (EMD Millipore)

  9. UNOSphere S cation exchange media (Bio-Rad).

  10. Kimble-Chase Kontes FlexColumn (Fisher).

  11. Pierce™ BCA Protein Assay Kit (ThermoFisher).

  12. Circular Dichroism quartz cuvette (Hellma)

  13. EnzChek® Lysozyme Assay Kit (ThermoFisher).

3. Method

3.1 Overexpression of HEWL in E. coli

All procedures are performed at room temperature under sterile conditions unless otherwise indicated.

  1. Transform a plasmid encoding HEWL into CaCl2 competent E. coli BL21(DE3) cells as follows: Add 2 μL of HEWL DNA to 75 μL of competent BL21(DE3) cells. Incubate the mixture on ice for 30 min. Heat shock the mixture at 42 °C for 45 s, and then incubate on ice for two min. To rescue the cells, add 250 μL of SOC media to the mixture, and incubate in an incubator shaker at 37 °C and 225 rpm. for one h. Remove from the shaker, and plate cells (150 μL) on an LB/Kanamycin agar plate. Incubate the plate for 10–12 h in an incubator at 37 °C.

  2. Pick a single colony of the transformed cells containing the HEWL vector into 10 mL of LB containing 10 μL of 40 mg/mL kanamycin sulfate (final concentration of 40 μg/mL). Incubate this mixture for 10–12 h in an incubator shaker at 37 °C and 225 rpm.

  3. Transfer this 10 mL solution into a 2 L baffled flask containing 1 L of LB and 1 mL of 40 mg/mL kanamycin sulfate (final concentration of 40 μg/mL). Grow the culture in an incubator shaker at 37 °C and 225 rpm until the cells reach an optical density with OD600 of 0.8 (≈2.5 h). Then, add 1 mL of the 1 M IPTG (final concentration of 1 mM) and incubate for another 6 h at 30 °C and 225 rpm.

  4. Centrifuge the culture at 5524 g for 20 min at 4 °C. Remove the supernatant and discard into 10% bleach. Re-suspend the pellet in Buffer 1 (20 mL) by mixing or pipetting the solution over the pellet, but do not vortex the pellet (see Note 5).

  5. Lyse the re-suspended cells using probe sonication. For this process, incubate the cells on ice, and use eight cycles of 30 s pulses (20 Watt) with 1 min of cooling (without sonication) between cycles (see Note 6).

  6. Centrifuge the viscous solution at 4 °C for 50 min at 30,996 g with a floor model centrifuge (eg. Beckman Avanti™ J-25) with a rotor suitable for 50 mL centrifuge tubes (eg. Beckman JA-17).

  7. Discard the supernatant and retain the pellet, which contains HEWL in inclusion bodies (see Note 7). Add 30 mL of Buffer 2 to the pellet, and incubate at 4 °C with shaking overnight to fully dissolve, denature, and solubilize any remaining protein (see Note 8).

  8. Centrifuge the dissolved pellet at 33,264 g for 1 h. Retain the supernatant and discard any remaining pelleted cell debris. The supernatant should now contain denatured HEWL.

  9. HEWL may then be purified using UNOSphere S-Cation exchange media in a column by hand. For this procedure, add 3 mL of the resin to a Kontes FlexColumn, and rinse several times with water (1.0 mL/min) and then 3–4 column volumes of Buffer 2 (1.0 mL/min). Incubate the denatured HEWL with the resin for 2 h at 4 °C, and then wash the resin-adhered protein with 50 mL of Buffer 2 (1.0 mL/min). Then elute the purified protein from the column using 50 mL of Buffer 3 (1.0 mL/min). SDS-PAGE can be used to determine the purity of the eluted HEWL (see Note 9).

  10. Protein concentration in the eluted solution is then measured by measuring the absorbance at 280 nm (molar extinction coefficient for HEWL of 37970 M−1 cm−1) or by using a BCA assay kit. The solution is then either concentrated or diluted (Buffer 3) to ≈4.4 mg/mL using a 10 kDa concentrator. This protein solution can next be folded.

3.2 Refolding HEWL using the VFD

The VFD should be set at an angle of 45° relative to the horizontal position and the bearings should be well oiled (see Note 10).

3.21 Small Scale Folding of HEWL by confined mode VFD processing (<3 mL)

  1. To lower the concentration of urea from the HEWL solution in Buffer 3, disperse 10 μL of the solution in 990 μL of Buffer 4, and mix with a 1 mL pipette.

  2. Add the diluted solution (≈44 μg/mL) to a 20 mm external diameter glass sample tube, and seal the sample tube with a Suba-Seal® septum.

  3. Insert the sample tube into the VFD. To do this, first apply a thin layer of lubricant around the sample tube. Then, insert the sample tube gently through the upper housing unit, until it is firmly mounted in the housing unit of the lower bearing.

  4. Place the safety shield on the device, and rotate the sample tube at a 5 krpm rotational speed for 5 min (see Note 11).

  5. Remove the contents of the tube, and transfer into a 2 mL centrifuge tube containing 100 μL of 100% glycerol. Quickly mix, and flash freeze the sample in liquid nitrogen or a mixture of dry ice and ethanol. Store the sample at −80 °C until analysis (see Note 12).

3.22 Large Scale Folding of HEWL by Continuous Flow VFD processing

  1. The solution of HEWL, currently in Buffer 3 is rapidly diluted into Buffer 4. Disperse 1 mL of the HEWL solution in Buffer 3 in 99 mL of Buffer 4 contained in a 250 mL beaker or flask. Mix this solution rapidly by swirling.

  2. As shown in Fig 2, the VFD can be configured for continuous flow operation. Process the solution by flowing through the VFD at a rate of 0.1 mL/min. Collect the solution of folded protein in a beaker or flask on ice.

Figure 2.

Figure 2

Open in a new tab

A step-by-step guide for continuous flow protein folding by VFD. A) Insert the 20 mm external diameter sample tube. For this process, we suggest a lubricant be added to the sample tube before applying any force. B) Insert a metal jet feed through the housing lid until ≈75% of the jet feed has been inserted (see Note 14). C) Add the housing lid onto the housing unit and fasten with plastic screws. Orient the jet feed with the exit facing the lower side of the sample tube. D) Draw the solution of unfolded HEWL into a 50 mL syringe, and vent any air bubbles. E) Insert the syringe first into the jet feed, and then place the syringe into the syringe pump. Select the required flow rate and start the VFD and syringe pump. For the confined mode of operation (1–3 mL), add the unfolded HEWL solution to the sample tube via a pipette, and cap with a Suba seal.

3.23 Analysis of Refolded HEWL

Circular Dichroism
  • 1

    Circular Dichroism (CD) is used to determine the secondary structure of the refolded HEWL samples. The sample (200 or 300 μL) is added directly to a CD cuvette. The spectrum is obtained using a spectropolarimeter such as the Jasco J-810 spectropolarimeter scanning 20 nm/min over four accumulations. Based on previous published results and crystal structures, the expected secondary structure of HEWL is largely α-helical (28) (see Note 13).

HEWL Activity Assay
  • 2

    To verify the protein folding efficacy of the sample, perform a HEWL activity assay. The experiment uses the commercially available Enzcheck Lysozyme Assay Kit. Follow the manufacturer instructions to determine the activity of the refolded samples. A non-VFD-processed sample provides a direct comparison.

Figure 1.

Figure 1

Open in a new tab

Schematic diagram of a VFD. A) The VFD and its rotational speed controller unit. The electric motor connects to the controller box and the lower bearing. Both the upper and lower bearings hold the sample tube in place during rotation. B) The sample tube is inserted into the upper bearing. C) The upper bearing of the device showing connection to the housing unit by six screws. D) The housing unit of the VFD. The holder to the continuous flow jet feeds, termed the housing lid, attaches to the housing unit via the screw inserts.

Footnotes

1

NaH2PO4 can be difficult to dissolve and requires thorough mixing. We recommend using a stir plate, and optionally gentle heating.

2

Add all components of the buffer, apart from the urea, to 300 mL of ddH2O as this large amount of urea will greatly increase the solution volume. The reaction is endothermic and will require gentle heating and stirring to completely dissolve the salts. The pH of this buffer is temperature dependent; thus, allow the solution to equilibrate at room temperature before adjusting the pH as detailed above.

3

As with Buffer 2, Buffer 3 requires heating with stirring. The pH should again be adjusted after allowing the solution to reach room temperature.

4

The bearings in the VFD need to be replaced every ≈3000 hours. See Figure 1 for bearing location during replacement.

5

At this point, the re-suspended solution can be stored at −80 °C after addition of 10% glycerol.

6

Other methods of lysing cells such as using a French press are also acceptable.

7

A layer of cell debris can often be observed on the outside of the pellet. This layer can be removed and discarded along with removal of the supernatant.

8

We find it easier to dissolve the pellet in urea by breaking up the pellet into smaller pieces using a sterile spatula. We then transfer the solution into a plastic conical tube to dissolve overnight.

9

If purity is poor after this step, wash steps can be added. Slowly increase the concentration of NaCl in the binding buffer from 0 mM to 50 mM in 10 mM increments. Additionally, size exclusion chromatography can be performed to further purify the protein.

10

As shown in several publications (25, 16), any deviation from the 45° tilt angle may drastically reduce the efficiency of the VFD. Additionally, the vibrations responsible for driving protein folding are produced by vibrations inherent to the VFD. Such vibrations can be sensitive to small changes in operational parameters, such as tilt angle, rotational speed, sample tube size, and wear on housing unit and bearings. Therefore, device maintenance is critical. For example, the addition of oil (e.g. machine oil, Singer) to the bearings before each use can increase reproducibility.

11

Several factors contribute to the optimal rotational speed for protein refolding, including quality and wear of both bearings and the housing unit. If refolding is not observed, vary the rotational speed of the sample tube in 50 rpm increments around 5 krpm to optimize folding conditions. Additionally, the housing unit and bearings can be replaced easily. The link below contains a 3D printing file for the housing unit (https://grabcad.com/library/vfd-collar-1, Fig. 1); high density ABS plastic is sufficient for production of this part.

12

Rapid dilution is commonly used to fold proteins. For this comparison between VFD and non-VFD processed conditions, samples are flash frozen after processing to prevent further conformational changes. For the non-VFD control, identical treatments include dilution of the sample and incubation at room temperature for the same time period as VFD processing (5 min); therefore, a direct comparison can be made with the VFD-processed solution.

13

Use the Dichroweb program to analyze the CD spectrum of the proteins, and assign its secondary structure (29).

14

The jet feeds must be held firmly in the housing unit lid. If they are not held firmly, then vibration of the jet feeds against the rotating sample tube will cause the glass sample tube to shatter.

References

  • 1.Andersen DC, Krummen L. Recombinant protein expression for therapeutic applications. Current Opinion in Biotechnology. 2002;13(2):117–123. doi: 10.1016/s0958-1669(02)00300-2. doi: http://dx.doi.org/10.1016/S0958-1669(02)00300-2. [DOI] [PubMed] [Google Scholar]
  • 2.Hunt I. From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expression and Purification. 2005;40(1):1–22. doi: 10.1016/j.pep.2004.10.018. doi: http://dx.doi.org/10.1016/j.pep.2004.10.018. [DOI] [PubMed] [Google Scholar]
  • 3.Graumann K, Premstaller A. Manufacturing of recombinant therapeutic proteins in microbial systems. Biotechnology journal. 2006;1(2):164–186. doi: 10.1002/biot.200500051. [DOI] [PubMed] [Google Scholar]
  • 4.Hewitt L, McDonnell JM. Screening and optimizing protein production in E. coli. Methods in molecular biology (Clifton, NJ) 2004;278:1–16. doi: 10.1385/1-59259-809-9:001. [DOI] [PubMed] [Google Scholar]
  • 5.Rosano GáL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology. 2014:5. doi: 10.3389/fmicb.2014.00172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Wang L, Maji SK, Sawaya MR, Eisenberg D, Riek R. Bacterial inclusion bodies contain amyloid-like structure. PLoS biology. 2008;6(8):e195. doi: 10.1371/journal.pbio.0060195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chang ESH, Liao T-Y, Lim T-S, Fann W, Chen RPY. A New Amyloid-Like β-Aggregate with Amyloid Characteristics, Except Fibril Morphology. Journal of Molecular Biology. 2009;385(4):1257–1265. doi: 10.1016/j.jmb.2008.11.009. doi: http://dx.doi.org/10.1016/j.jmb.2008.11.009. [DOI] [PubMed] [Google Scholar]
  • 8.Ventura S, Villaverde A. Protein quality in bacterial inclusion bodies. Trends in Biotechnology. 2006;24(4):179–185. doi: 10.1016/j.tibtech.2006.02.007. doi: http://dx.doi.org/10.1016/j.tibtech.2006.02.007. [DOI] [PubMed] [Google Scholar]
  • 9.Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Factories. 2015:14. doi: 10.1186/s12934-015-0222-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Vallejo LF, Rinas U. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories. 2004;3:11. doi: 10.1186/1475-2859-3-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Qoronfleh MW, Hesterberg LK, Seefeldt MB. Confronting high-throughput protein refolding using high pressure and solution screens. Protein Expression and Purification. 2007;55(2):209–224. doi: 10.1016/j.pep.2007.05.014. doi: http://dx.doi.org/10.1016/j.pep.2007.05.014. [DOI] [PubMed] [Google Scholar]
  • 12.Radford SE. Protein folding: progress made and promises ahead. Trends in biochemical sciences. 2000;25(12):611–618. doi: 10.1016/s0968-0004(00)01707-2. [DOI] [PubMed] [Google Scholar]
  • 13.Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif. 2003;28(1):1–8. doi: 10.1016/s1046-5928(02)00641-1. [DOI] [PubMed] [Google Scholar]
  • 14.Peng W, Chen X, Zhu S, Guo C, Raston CL. Room temperature vortex fluidic synthesis of monodispersed amorphous proto-vaterite. Chemical Communications. 2014;50(79):11764–11767. doi: 10.1039/C4CC05607H. [DOI] [PubMed] [Google Scholar]
  • 15.Wahid MH, Eroglu E, LaVars SM, Newton K, Gibson CT, Stroeher UH, Chen X, Boulos RA, Raston CL, Harmer S-L. Microencapsulation of bacterial strains in graphene oxide nano-sheets using vortex fluidics. RSC Advances. 2015;5(47):37424–37430. doi: 10.1039/C5RA04415D. [DOI] [Google Scholar]
  • 16.Yasmin L, Chen X, Stubbs KA, Raston CL. Optimising a vortex fluidic device for controlling chemical reactivity and selectivity. Scientific Reports. 2013;3:2282. doi: 10.1038/srep02282. http://www.nature.com/articles/srep02282 - supplementary-information. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Yasmin L, Eggers PK, Skelton BW, Stubbs KA, Raston CL. Thin film microfluidic synthesis of fluorescent highly substituted pyridines. Green Chemistry. 2014;16(7):3450–3453. doi: 10.1039/C4GC00881B. [DOI] [Google Scholar]
  • 18.Britton J, Chalker JM, Raston CL. Rapid Vortex Fluidics: Continuous Flow Synthesis of Amides and Local Anesthetic Lidocaine. Chemistry – A European Journal. 2015;21(30):10660–10665. doi: 10.1002/chem.201501785. [DOI] [PubMed] [Google Scholar]
  • 19.Britton J, Raston CL. Rapid high conversion of high free fatty acid feedstock into biodiesel using continuous flow vortex fluidics. RSC Advances. 2015;5(3):2276–2280. doi: 10.1039/C4RA14909B. [DOI] [Google Scholar]
  • 20.Gandy MN, Raston CL, Stubbs KA. Photoredox catalysis under shear using thin film vortex microfluidics. Chemical Communications. 2015;51(55):11041–11044. doi: 10.1039/C5CC02153G. [DOI] [PubMed] [Google Scholar]
  • 21.Mo J, Eggers PK, Chen X, Ahamed MRH, Becker T, Yong Lim L, Raston CL. Shear induced carboplatin binding within the cavity of a phospholipid mimic for increased anticancer efficacy. Scientific Reports. 2015;5:10414. doi: 10.1038/srep10414. http://www.nature.com/articles/srep10414 - supplementary-information. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Vimalanathan K, Gascooke JR, Suarez-Martinez I, Marks N, Kumari H, Garvey CJ, Atwood JL, Lawrance WD, Raston CL. Fluid dynamic lateral slicing of high tensile strength carbon nanotubes. Scientific Reports. 2016;6:22865. doi: 10.1038/srep22865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yasmin L, Stubbs KA, Raston CL. Vortex fluidic promoted Diels–Alder reactions in an aqueous medium. Tetrahedron Letters. 2014;55(14):2246–2248. doi: http://dx.doi.org/10.1016/j.tetlet.2014.02.077. [Google Scholar]
  • 24.Britton J, Raston CL. Continuous flow vortex fluidic production of biodiesel. RSC Advances. 2014;4(91):49850–49854. doi: 10.1039/C4RA10317C. [DOI] [Google Scholar]
  • 25.Britton J, Dalziel SB, Raston CL. Continuous flow Fischer esterifications harnessing vibrational-coupled thin film fluidics. RSC Advances. 2015;5(3):1655–1660. doi: 10.1039/C4RA11777H. [DOI] [Google Scholar]
  • 26.Britton J, Dalziel SB, Raston CL. The synthesis of di-carboxylate esters using continuous flow vortex fluidics. Green Chemistry. 2016 doi: 10.1039/C5GC02494C. [DOI] [Google Scholar]
  • 27.Yuan TZ, Ormonde CFG, Kudlacek ST, Kunche S, Smith JN, Brown WA, Pugliese KM, Olsen TJ, Iftikhar M, Raston CL, Weiss GA. Shear-Stress-Mediated Refolding of Proteins from Aggregates and Inclusion Bodies. ChemBioChem. 2015;16(3):393–396. doi: 10.1002/cbic.201402427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Tanaka FF, Leslie Pai, Pranab K, Rupley John A. The Circular Dichroism of Lysozyme. The Journal of Biological Chemistry. 1975;250(17):6977–6982. [PubMed] [Google Scholar]
  • 29.Whitmore L, Wallace BA. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Research. 2004;32(Web Server issue):W668–673. doi: 10.1093/nar/gkh371. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES