Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

The goal of this experiment is to replace a canonical amino acid by a non-canonical amino acid for recombinant protein production by selective pressure incorporation to produce the novel gold fluorescent protein. The method of selective pressure incorporation is intended to produce proteins with novel chemical features. This is especially useful when attempting to understand or to engineer protein function.

The main advantage of this technique is that it is relatively simple to conduct because it similar to common recombinant protein production. Time-and wavelength-correlated fluorescence is helpful to investigate the excited state dynamics of the modified protein, which in turns makes it easier to understand the influence of the non-canonical amino acid introduced into the chromophore on the optical properties. SPI can, in principle, be applied to any protein production setting for which auxotrophic strains and chemically defined media are available.

This includes other bacterial strains and also mammalian cell culture. The timing and the setup of expression conditions are critical since it's mandatory to suppress the production of the non-modified wild-type protein. That's why certain parameters such as the temperature, the time of induction, or the composition of the cultivation medium need to be optimized.

We first had the idea for this method after initial studies of protein extract with methyl-containing amino acids such as selenomethionine. Demonstrating the procedure will be Almut Pelzer, a student from my laboratory. To begin this procedure, transform tryptophan auxotrophic E.coli cells with the expression plasmid for ECFP.

Use a single colony to inoculate a preculture of five milliliters LB medium supplemented with ampicillin in a culture tube. Incubate overnight at 37 degrees Celsius and 200 rpm. The next day, inoculate 100 milliliters of supplemented NMM19 medium using one milliliter of the overnight culture in a one-liter Erlenmeyer flask.

Then, incubate the culture overnight in an orbital shaker at 200 rpm and 30 degrees Celsius. The next day, measure the optical density at 600 nanometers every 30 minutes until the value changes by less than 0.05 over 30 minutes. Next, centrifuge the culture at 5, 000 times g and four degrees Celsius for 10 minutes to harvest the bacterial cells.

Discard the supernatant by decanting. Resuspend the cells in NMM19 medium supplemented with ampicillin to a total volume of 100 milliliters. Transfer this suspension back to the same Erlenmeyer flask.

Add 4-amino-indole to a final concentration of one millimolar. Incubate in an orbital shaker for 30 minutes at 30 degrees Celsius and 200 rpm. After 30 minutes, add IPTG to the culture to a final concentration of 0.5 millimolar to induce target gene expression.

Incubate overnight in an orbital shaker at 30 degrees Celsius and 200 rpm. The next day, harvest the bacterial cells by centrifuging at 5, 000 times g and four degrees Celsius for 10 minutes. Discard the supernatant by decanting.

Freeze the cell pellet at minus 20 degrees Celsius or minus 80 degrees Celsius until ready to perform target protein purification. First, prepare the bacterial cell lysate as outlined in the text protocol. After preparing the chromatography column according to the manufacturer's instructions, use binding buffer to equilibrate the immobilized metal ion affinity chromatography column.

Load the lysate on the column until completion. Notice the gold-colored protein bound to the chromatography resin. Wash the column with binding buffer.

Then, elute the target protein using elution buffer. Collect and pool any eluted fractions that contain gold fluorescent protein, which can be identified by the visible golden color. After this, prepare a dialysis membrane with a molecular weight cutoff between 5, 000 and 10, 000 according to the manufacturer's instructions.

Dialyze a one-milliliter sample against at least 100 milliliters of buffer for a minimum of two hours. To begin, use MS buffer to dilute the protein sample to 0.1 milligrams per milliliter, such that the final volume is 80 microliters. Pipette carefully to mix.

Transfer the solution to an MS autosampler vial with glass insert, and close it with a cap. Flick the vial to remove air bubbles. Next, prepare a blank containing MS buffer in a second vial.

Put both vials into the HPLC autosampler. After this, prepare and calibrate the MS instrument according to the manufacturer's instructions. Set the autosampler injection volume to five microliters for the high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry method.

Create a work list for the blank run, followed by the GdFP sample run. Assign the injection volume, the corresponding autosampler vial positions, and run the work list. To begin, prepare a gold fluorescent protein solution and the detector for time-and wavelength-correlated single photon counting as outlined in the text protocol.

Using single photon counting software, acquire fluorescence emission at a count rate of about 200, 000 photons per second until approximately 10, 000 counts are accumulated in the acquisition maximum of the fluorescence decay curves. After this, replace the the sample cuvette with a one-centimeter quartz cuvette filled with colloidal silica in PBS buffer. Take out the laser filter, and adjust the grating for the acquisition of 470-nanometer photons in the central channel of the detector.

Then, acquire the instrumental response function until approximately 10, 000 counts are accumulated in the emission maximum. Convert, fit, and plot the resulting data as outlined in the text protocol to obtain the decay-associated spectra, which represents the dependence of the fluorescence decay times on the emission wavelength. In this study, mass spectrometry is used to confirm that the spectrally red-shifted green fluorescent protein-type fluorophore variant, termed gold fluorescent protein, is produced successfully.

While wild-type ECFP has a calculated protein mass of 28, 283.9 daltons after the chromophore maturation, the corresponding mass of gold fluorescent protein is 28, 313.9 daltons. The deconvoluted ESI-MS spectrum of gold fluorescent protein exhibits a main mass peak at approximately 28, 314.1 daltons, deviating from the theoretical value by less than 10 parts per million. This confirms the incorporation of the non-canonical amino acid by selective pressure incorporation.

The absorption spectrum of ECFP has two characteristic maxima at 434 nanometers and 452 nanometers. In contrast, gold fluorescent protein is characterized by one broad red-shifted absorption band with the maximum at 466 nanometers. While the absorption maximum of EGFP is further red-shifted to 488 nanometers, the fluorescence emission spectrum of gold fluorescent protein and the corresponding Stokes shift of GdFP signifies that it is the most red-shifted of all the derivatives of Aequorea victoria green fluorescent protein.

The time-resolved fluorescence emission monitored by single photon counting is shown here. The decay curves exhibit a slightly faster fluorescence decay at 600 nanometers than at 550 nanometers. The fluorescence emission of gold fluorescent protein strongly depends on pH, as is typical for many green fluorescent protein variants.

As seen here, there is a clear decrease in the fluorescence at lower pH, though the spectral characteristics stay constant. Once mastered, this technique can be done in three days when starting from colonies from transformed bacterial cells or from frozen glycerol stocks. While attempting this procedure, it is important to ensure that the targeted amino acid is depleted.

The ncAA must be taken up by the cell, biocompatible, and incorporated into proteins by the chosen auxotrophic strain. Following this procedure, a large variety of proteins can be generated for engineering purposes. Depending on the target protein methods, like protein stability analyzers, enzymatic activity assays, fluorescence spectroscopy, or protein crystallization, can be used to describe the influence of the non-canonical amino acid on functional and structural properties.

After its development, this technique proved to be very useful in the field of synthetic biology, providing it with new-to-nature proteins and proteomes. This expands the chemical capacities for protein engineering. After watching this video, you should have a good understanding of how to incorporate non-canonical amino acids into recombinant proteins by selective pressure incorporation, how to verify the introduction of an artificial amino acid by mass spectrometry, and how to analyze a fluorescent protein by time-resolved fluorescence.