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

The scope of this research asks whether solvent extraction precipitation can rapidly purify ELP fusion proteins while retaining fusion activity function. Recent developments include rapid solvent-based ELP purification with improved endotoxin control, enabling active self-assembling nanoparticles for targeted nucleic acid delivery. To begin, weigh out one gram of the E.coli cell pellet.

Add four milliliters of freshly prepared lysis buffer to resuspend the cell pellet. Vortex gently until the pellet is fully dispersed and mixed with the buffer. Add approximately five milligrams of lysozyme to the resuspended cells to assist in cell wall digestion.

Then place the tube on ice and incubate for one hour. Following incubation, sonicate the sample using a micro-tip probe sonicater in five-second intervals. Keep the sample on ice and continue sonication until a uniform lysate free of visible particulates is obtained typically for five to eight minutes.

Centrifuge the sonicate lysate at 10, 000 G for 10 minutes at 20 degrees Celsius to clarify it. Then carefully separate the supernatant containing the soluble fraction from the pellet containing the insoluble fraction. Pipette out 10 to 20 microliters of the supernatant and resuspend an equivalent mass of the pellet in lysis buffer.

Mix both fractions with 4x Laemmli buffer to a final 1x concentration. Then heat the suspension at 95 degrees Celsius for five minutes. If the ELP is predominantly soluble, proceed with organic solvent extraction using the clarified supernatant.

To perform solvent extraction, add four milliliters of organic solvent or solvent mixture to the air-dried pellet or supernatant. For binary solvent mixtures, use exact volumetric ratios, such as two milliliters of each for a one-to-one combination. Vortex the suspension vigorously for one minute to ensure proper solvent penetration into the pellet.

Now, incubate the mixture at 20 degrees Celsius for five minutes to allow aggregation of extracellular proteins and solubilization of ELP in the organic phase. Centrifuge the sample at 13, 000 G for 10 minutes at 20 degrees Celsius to separate solubilized proteins from insoluble debris. Carefully collect the supernatant without disturbing the pellet as it contains the solubilized ELP.

Measure the volume of the collected supernatant precisely to prepare for precipitation. Next, add acetone or acetonitrile to the supernatant at 2.33 times its volume for protein precipitation. Incubate the mixture at 20 degrees Celsius for five to seven minutes.

Centrifuge the sample at 10, 000 G for 10 minutes at 20 degrees Celsius. Then discard the supernatant and air dry the pellet either under a gentle nitrogen gas stream for 10 to 15 minutes or by placing uncapped centrifuge tubes upside down on lint-free wipes in a fume hood for one hour at 20 degrees Celsius. Resuspend the dried protein pellet in 50 microliters of PBS.

Then pipette up and down gently to ensure complete dissolution. Store the resuspended protein at minus 20 degrees Celsius for short-to-medium term use. To perform validation using SDS-PAGE, first mix the purified protein with 4x SDS-PAGE loading buffer.

Vortex the solution briefly for homogeneity. Load the sample onto a 10%SDS-PAGE gel for ELP and TEEN fused to chorismate mutase. Run the gel at 100 to 120 volts until the tracking dive reaches the bottom.

Now, stain the gel using InstantBlue Coomassie stain or a suitable alternative for two hours at 20 degrees Celsius. Rinse with deionized water until a clear background is achieved. A prominent ELP and TEEN fused to chorismate mutase band appeared at 100 kilodaltons following a lysis step prior to organic extraction.

Different organic solvent combinations resulted in varied extraction efficiencies and purities of ELP and TEEN fused to chorismate mutase. The AG and BG one-to-one mixtures yielded the highest protein recovery. The AG solvent extracted ELP and TEEN fused to chorismate mutase retained approximately 80%of its enzymatic activity compared to ITC-purified protein, while BG retained around 50%activity, and the V40 control showed almost no activity.

Significant findings include demonstrating that organic solvent extraction precipitation rapidly purifies ELP fusions with low endotoxin while preserving fusion protein activity. This protocol addresses the lack of rapid detergent-free methods to purify ELP fusions from E.Coli with or without inclusion bodies without multi-step ITC. This protocol offers a single-step rapid purification directly from E.coli pellets, yielding highly pure, low-endotoxin ELP fusions while preserving fusion protein function.