Efficient Purification of Elastin-Like Polypeptides (ELPs) from E. coli Using an Organic Solvent-based Extraction and Precipitation Method

Summary

This protocol describes a rapid, reproducible organic solvent-based extraction and precipitation method for purifying elastin-like polypeptide (ELP) from Escherichia coli, providing a potentially scalable alternative to conventional ELP purification methods.

Abstract

Elastin-like polypeptides (ELP) are engineered biopolymers built from repetitive pentapeptide sequences that mimic motifs found in mammalian tropoelastin. Their unique characteristics make them ideal candidates for a wide array of biomedical applications, ranging from drug and gene delivery to tissue engineering and targeted molecular imaging. Conventional purification approaches from Escherichia coli (E. coli) expression can be ineffective for ELP due to the formation of inclusion bodies. Other methods, such as inverse transition cycling (ITC), utilize the lower critical solution temperature (LCST) properties of ELP to separate it from contaminants such as lipopolysaccharides (LPS), but typically require multiple heating and cooling steps that are time-consuming and can result in low recoveries depending on the sequence, concentration, and molecular weight of the ELP construct. To tackle these challenges, we have developed an organic solvent-based extraction-precipitation workflow that exploits the intrinsic hydrophobicity of ELP to enable rapid, robust, and broadly applicable purification directly from E. coli cell pellets. This method uses polar organic solvents to aid in cell disruption and selectively solubilize ELP in a single step. A subsequent precipitation step effectively removes residual organic solvents, low-molecular-weight impurities, and endotoxins, yielding highly pure ELP with LPS levels below 1 EU/mL in under 3 h. Atomic force microscopy data suggest that ELP-fusion proteins purified in this manner can self-assemble into reverse micelle-like structures that retain fusion protein function. This rapid purification method offers researchers a straightforward and potentially scalable way to purify ELP, creating new possibilities for using ELP and their fusion proteins as flexible building blocks for material and biomedical applications.

Introduction

Elastin-like polypeptides (ELPs) are biopolymers composed of repeating (VPGXG)n motifs^{1,2,3,4,5,6,7,8,9}. The guest residue X in the pentapeptide unit can be any amino acid other than proline. Variations in X can be used to tune the hydrophobicity, thermal transition behavior, and functional characteristics of the ELP construct to create modular biocompatible materials that have been widely explored in various biomedical applications^10,11,12 .

While chemical synthesis can be used to produce short peptides, it is poorly suited for high molecular weight ELPs or for retaining the functionality of fusion proteins^13,14. For reasons such as these, ELPs are typically expressed recombinantly in E. coli, allowing for sequence-defined production in large quantities. However, this approach can also bring challenges. During their expression in E. coli, ELPs often accumulate within dense inclusion bodies, requiring efficient recovery strategies to extract functional protein¹⁵. Moreover, bacterial expression introduces unwanted biomolecules such as host cell proteins, nucleic acids, and lipopolysaccharides (endotoxins) that must be separated from the ELP during purification. Traditional solubilization strategies using detergents (e.g., sodium dodecyl sulphate (SDS) or Triton X-100) or chaotropic agents like urea have met with limited success for our ELP fusion sequences. In our experience, Triton and urea failed to extract ELPs from the pellet phase, while SDS enabled partial solubilization but interfered with downstream affinity purification and was difficult to fully remove from the hydrophobic ELP sequence.

To date, inverse transition cycling (ITC) remains the most widely used purification method for ELPs. This technique exploits their LCST behavior to selectively precipitate and resolubilize the ELP across temperature and salt gradients¹⁶. However, with lower molecular weight proteins, ITC is a time-consuming multi-step process that is poorly suited for ELPs trapped in inclusion bodies, requiring multiple ITC cycles that can lead to protein loss at each step^7,16.

To overcome these limitations, we adopted an organic solvent-based extraction and precipitation workflow. While Yakhnin et al. first demonstrated a hydrophobicity-driven partitioning strategy for GFP purification using ethanol and salt gradients in 1998¹⁷, the novel method we developed employs organic solvent blends to directly extract ELP fusion proteins from E. coli cells⁷. Sweet et al. and Darji et al. adapted this approach to ELP fusions, showing that organic solvent extraction enables rapid recovery of functional ELPs while maintaining structural integrity and bioactivity^8,9. This approach uses sonication and organic solvents to lyse bacterial cells and extract ELPs in a single operation, while precipitating most of the host proteins and nucleic acids. Recovery of the ELP-rich organic phase, followed by rapid precipitation to isolate the ELPs from solvents and solubilized impurities, yields highly pure ELPs. This approach is also capable of yielding pure ELP from E. coli pellets with endotoxin levels below 1 EU/mL in under three hours^7,8.

Studies to date show that this method is capable of purifying ELP constructs and ELP fusion proteins of varying molecular weight and isoelectric point, without multiple ITC cycles or solubilization steps (Figure 1). Atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging suggest that ELP-fusion proteins purified in this manner form reverse micelle-like structures (Figure 2), thus helping to preserve the structure and function of ELP fusion domains during organic solvent extraction. This streamlined workflow enables faster and more reliable ELP purification for diverse biomedical applications^7,8. Notably, Aayush et al. reported that ELP constructs purified by this method retained epidermal growth factor receptor-binding function¹⁸, highlighting the point that this method not only produces pure ELP material, but also maintains the activity of the fusion protein domain.

As a further demonstration of the utility of this method in isolating an ELP-enzyme fusion, we describe a detailed organic solvent-based method to purify an ELP-Intein-Chorismate mutase 2 (ELP-I-Cm2) construct (Figure 3). This method may be useful for rapid purification of other ELPs for drug delivery and nanomaterials applications.

Protocol

1. Materials preparation

1. Bacterial strain: Use Escherichia coli BL21(DE3) chemically competent cells as the expression host.
   NOTE: They were selected due to their high transformation efficiency and suitability for recombinant protein production.
2. Expression plasmid: Use pET21(+) vector encoding ELP-I-Cm2 fusion protein with ampicillin-resistance⁹. Purchase the plasmid vector from addgene. Insert the Cm2 gene using the BsrG I site at the end of the intein.
   NOTE: The plasmid DNA sequence for this protein is provided in Supplementary File 1.
3. Growth media: Terrific Broth (TB) media preparation for 1200 mL media
   1. Prepare media (Part 1) by adding 16 g of Tryptone, 32 g of Yeast Extract, 16 g of proline, 6.8 mL of glycerol to 1100 mL of ultrapure water. Mix thoroughly to dissolve all components. Adjust the final volume to 1200 mL using ultrapure water.
   2. Prepare the potassium phosphate buffer (Part 2) by mixing 23.1 g of KH₂PO₄ and 125.4 g of K₂HPO₄, and then bring the volume to 1 L with ultrapure water.
   3. Autoclave the components (Parts 1 and 2) separately. After autoclaving, assemble the media by dividing the TB base into four 300 mL aliquots. Add 33 mL of phosphate buffer to each aliquot under sterile conditions.
4. Additives for expression
   1. IPTG: Perform induction using IPTG at a final concentration of 1.2 mM (e.g., add 396 µL of 1 M IPTG to 330 mL culture).
   2. Antibiotic: Add ampicillin to a final concentration of 100 µg/mL to ensure plasmid selection.
5. Lysis buffer (pH 8.5): Prepare the lysis buffer with 10 mM Tris Base and 22 mM ethylenediaminetetraacetic acid (EDTA). Adjust the pH to 8.5 using HCl or NaOH as required.
6. Organic solvents for extraction
   1. Employ a variety of organic solvents (Table 1) to extract the ELP-I-Cm2 fusion protein from inclusion bodies. Test these solvents both individually and in binary combinations (1:1 vol: vol) to evaluate efficiency and ELP purity.
      NOTE: All solvents used were HPLC Grade, except Acetonitrile (Optima grade), Ethanol (USP grade), and 1-Butanol (99% pure).
      CAUTION: All solvents listed above are volatile and flammable. They must be handled inside a chemical fume hood using appropriate PPE (lab coat, nitrile gloves, and eye protection). Many types of plasticware are not compatible with organic solvents. Polypropylene plasticware is highly recommended for extraction-precipitation experiments with organic solvents; otherwise, the plasticware to be used should be tested without a valuable sample to ensure that it is appropriate for use. The disposal of solvent waste should follow the procedures outlined in the relevant institutional hazardous waste policies.

2. Cell lysis and preparation (Figure 4A)

1. Resuspension: Weigh out 1 g of cell pellet and resuspend it in 4 mL of freshly prepared lysis buffer. Vortex gently until the pellet is fully dispersed and mixed with the buffer.
2. Enzymatic lysis: Add ~5 mg of lysozyme to assist in cell wall digestion. Allow the suspension to incubate at 4 °C for 1 h. This facilitates partial enzymatic digestion to boost cell lysis.
3. Sonication: After incubation, sonicate the sample to complete cell lysis and shear genomic DNA. Use short bursts of microtip probe sonication (350 W device, power level 3) with intervals to avoid overheating (e.g., 5 s on/10 s off, repeated as needed). Sonicate the samples on ice and apply the ultrasonic energy until a uniform lysate free of visible particulates is obtained, typically for about 5-8 min.
   NOTE: After sonication is complete, store a 100 μL aliquot of the sample (or the lysate) for ELP expression analysis to monitor purification progress before and after the extraction and precipitation steps.
4. Centrifugation and expression analysis.
   1. Clarify the lysate by centrifugation at 10,000 g for 10 min at 20 °C, then carefully separate the supernatant (soluble fraction) from the pellet (insoluble/inclusion-body fraction).
   2. Assess ELP expression in both fractions by SDS-PAGE: withdraw an aliquot (e.g., 10-20 µL) of the supernatant and resuspend an equivalent pellet mass in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA) to the same volume; mix each with 4x Laemmli buffer to a final 1x, heat at 95 °C for 5 min, and load equal amounts to enable direct comparison.
   3. If the target ELP is predominantly soluble, proceed with the organic-solvent extraction using the clarified supernatant. If it is predominantly insoluble (inclusion bodies), discard the supernatant and continue with the inclusion-body workflow (washing/solubilization and extraction) by processing the pellet by organic solvent extraction.
5. Pellet washing and drying (only for ELP in inclusion bodies): Invert the centrifuge tubes to allow gravity-assisted drainage of any residual lysis buffer. Once visible liquid has drained (~1-2 min), leave the pellet to air dry at room temperature for 5-10 min, ensuring it is free of excess moisture before proceeding to organic extraction.

3. Organic extraction (Figure 4B)

NOTE: Each ELP has its own preferred solvent condition for yielding high-purity extractions (e.g., AC performance in Figure 1). Therefore, an initial screen of 28 organic solvents and their combinations should be tested, and the isolates analyzed by PAGE to identify the optimal solubilization efficiency conditions for the construct of interest.

1. Solvent screening. Screen the individual solvents shown in step 1.6 and their 1:1 (v/v) binary solvent mixtures. Evaluate additional mixtures (e.g., 1:2, 2:1, 4:1) to enhance the effectiveness of the process.
   NOTE: At present, there are no guidelines for rationally selecting the most effective solvents or combinations, so a screening campaign is needed to select from solvents that have previously met with success in solubilizing hydrophobic protein aggregates. The final selection of the organic solvent extractant to be used for purification should be based on SDS-PAGE analysis of the isolates obtained from this screen.
2. Extraction procedure
   1. Solvent addition: Add 4 mL of organic solvent or solvent mixture to the air-dried pellet/supernatant obtained after lysis. For mixed combinations, ensure precise volumetric ratios (e.g., 2 mL of each for a 1:1 combination).
   2. Vortexing: Vortex the suspension vigorously for 1 min to ensure thorough mixing and penetration of solvent into the pellet.
   3. Incubation (Retention time): Allow the suspension to incubate at 20 °C for 5 min, ensuring sufficient interaction time for extracellular proteins to aggregate and sediment while the ELPs solubilize in the organic phase.
   4. Centrifugation: Centrifuge the mixture at 13,000 × g for 10 min at 20 °C to separate solubilized proteins from residual insoluble debris.
   5. Supernatant collection: Carefully collect the supernatant without disturbing the pellet. The supernatant contains the solubilized ELP, while the pellet contains host cell proteins, nucleic acids, and lipids.
   6. Volume measurement: Record the volume of the recovered supernatant precisely. Care should be taken to ensure the supernatant is free from any pellet particles. This measurement is critical for the downstream precipitation step.

4. Protein precipitation

1. Anti-solvent addition: Add acetone or acetonitrile to the organic supernatant at 2.33 times the measured volume of the supernatant.
2. Incubation: Incubate the mixture at 20 °C for 5-7 min.
3. Centrifugation: Centrifuge at 10,000 g for 10 min at 20 °C.
4. Pellet handling: After centrifugation, carefully discard the supernatant and air dry the sample by leaving the tube uncapped under a gentle nitrogen gas stream for 10-15 min, or incubate by placing uncapped centrifuge tubes upside down on lint-free wipes in a fume hood for 1 h at 20 °C (do not apply heat).

5. Protein pellet handling and resuspension

1. Resuspension: Following precipitation and air-drying, resuspend the protein pellet in 50 µL of phosphate-buffered saline (PBS). Gently pipette up and down to ensure the pellet is fully dissolved.
2. Storage: Store the resuspended protein at -20 °C for short- to medium-term use. Avoid repeated freeze-thaw cycles to maintain protein functional integrity.

6. Validation via SDS-PAGE

1. Sample preparation: Mix the purified protein with 4× SDS-PAGE loading buffer in a 3:1 ratio and vortex briefly to ensure homogeneity.
2. Electrophoresis conditions: Load onto a 15% SDS-PAGE gel for ELP-I-Cm2 and run at 100-120 V until the tracking dye reaches the bottom.
   NOTE: The % crosslinking in the gel should be based on the size of the ELP construct of interest
3. Staining and visualization: Stain the gel using Instant Blue Coomassie stain or other suitable stain for 2 h at 20 °C. Destain by rinsing with deionized water until a clear background is achieved. Test the protein isolated in activity assays to validate that activity retention has occurred after the organic solvent extraction-precipitation purification workflow.

Results

After going through the final phases of the protocol, the next step is visualization of the extraction-precipitation outcome by SDS-PAGE analysis and Coomassie blue staining. When there is a lysis step prior to organic extraction, a prominent ELP band should be apparent. In the case of ELP-I-Cm2, this band appears at 100 kDa (Figure 3A).

Screening of different organic solvent combinations demonstrated variable extraction efficiencies (Figure 3...

Discussion

ITC has previously been used for non-chromatographic purification of ELP¹⁹; however, it can be a time-consuming method and low-yielding process. In the methods detailed above, we describe an approach for the rapid purification of an ELP-I-Cm2 fusion protein, demonstrating how the organic solvent-based extraction-precipitation method can be effectively used for the purification of a wide variety of ELP and ELP fusions. The organic solvent protocol is not only faster than ITC, but also much more eff...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Bio-Rad	161-0710	SDS-PAGE Component
30% Acrylamide/Bis-acrylamide	TCI	A3218	SDS-PAGE Component
Acetone	Fisher Scientific	A949	Organic Solvent
Acetonitrile	Fisher Scientific	A996	Organic Solvent
Ampicilin	Gold Bio- Gold Biotechnology	A-301-25	Antibiotic
APS (Ammonium Persulfate)	Bio-Rad	1610700	SDS-PAGE Component
Butanol	Fisher Scientific	L13171	Organic Solvent
EDTA	Fisher Bioreagents	BP118-500	Lysis Buffer Component
Ethanol	Decon Labs	UN1170	Organic Solvent
Ethyl Acetate	Fisher Scientific	E195	Organic Solvent
Glycerol	Research Products International	G22020-0.5	Terrific Broth (TB) Media Component
Hydrochloric Acid	Fisher Scientific	A144SI-212	Lysis Buffer Component
Instant Coomasie stain	Abcam	ab119211	SDS-PAGE Component
IPTG	Gold Bio- Gold Biotechnology	12481C25	Protein Expression Component
Isopropanol	Fisher Scientific	A451-1	Organic Solvent
K2HPO4 (Potassium Phosphate Monobasic)	Wards' Science	470302-246	Part 2 Buffer Component
KH2PO4 (Potasium Phosphate Dibasic)	Wards' Science	470302-254	Part 2 Buffer Component
L-Proline	Research Products International	P5200-500.0	Terrific Broth (TB) Media Component
Lysozyme	Gold Bio- Gold Biotechnology	L-040-1	Lysis Buffer Component
Methanol	Fisher Scientific	A452	Organic Solvent
Native Sample Buffer	Bio-Rad	161-0738	SDS-PAGE Component
Protein Ladder	Gold Bio- Gold Biotechnology	P008-500	SDS-PAGE Component
Resolving Gel Buffer	Bio-Rad	1610798	SDS-PAGE Component
Sodim Dodecyl Sulfate (SDS)	Thermo Scientific	J18220-36	SDS-PAGE Component
Sodium Hydroxide	Fisher Scientific	S318-500	Lysis Buffer Component
Stacking Gel Buffer	Bio-Rad	1610799	SDS-PAGE Component
TEMED	Bio-Rad	1610801	SDS-PAGE Component
Tris Base	Fisher Bioreagents	BP-152	Lysis Buffer Component
Tryptone	Research Products International	T60065-1000.0	Terrific Broth (TB) Media Component
Yeast Extract	Research Products International	Y20025-1000.0	Terrific Broth (TB) Media Component