Understanding Stability of sfGFP Through Purification and

Refolding of Protein Under Exposure to Denaturants

Biol-272: Biochemistry Lab
Ibrahim Waheed
Professor Jennifer Herrera
October 15th, 2023
 Abstract

Superfolder GFP (sfGFP) is a powerful research tool because it allows for a more robust

application of its non-mutated predecessor, GFP. This variant increases the effectiveness of the

staple tag molecule by being brighter, more tolerant to mutation, and less likely to degrade. This

allows for sfGFP to be incorporated into molecules that the base form GFP would be unable to.

Our goal was to grow and purify our own sfGFP samples, and then characterize some of their

different properties to determine the stability of this protein variant. To grow our proteins, we

introduced plasmids containing sfGFP into E. coli cells, cultured the cells until the proteins

matured, and then lysed the cells to remove the sfGFP. We then purified our samples using an

affinity-chromatography protocol to isolate the sfGFP from any remaining cell material, and then

assessed the quality of our purification by running an SDS-PAGE gel to track the presence of

sfGFP in samples taken throughout our experimental timeline. We determined that the protein

was successfully purified, and then tested the refolding rate of our sfGFP when exposed to the

chemical denaturant urea and the thermal denaturation rate when exposed to heat to determine

the overall stability of the protein. We determined that sfGFP folding became unfavorable when

concentration of urea exceeded 2.7M, and when temperature exceeded 335 degrees kelvin.

Using our denaturation data, we were also able to estimate the Delta G for sfGFP folding in the

absence of urea to be -14.9 kj/mol.

Introduction

GFP was first discovered in 1962 by Osamu Shimomura in the jellyfish species Aequorea

victoria. This protein is found in the bell edge of the jellyfish, and is responsible for the

luminescent appearance we associate with the animal. The protein itself is barrel shaped,

consisting of 11 beta sheets wrapped around a central alpha helix (Figure 1). This central helix

houses the protein’s intrinsic chromophore, a region containing modified adjacent serine,
tyrosine, and glycine residues that are responsible for emitting fluorescence 1. In lab, we are

able to use light at wavelengths between 395 and 475 nm to induce fluorescence, specifically by

causing cyclization and oxidation of the three residues above to occur. The structure of GFP

gives it many of the properties that make it such a widely applicable tag molecule; it is relatively

non toxic to cells, stable over a broad pH range, resistant to heat and detergents, does not

require the presence or assistance of any cofactors to fluoresce, and continues to fluoresce

even after being stimulated for a relatively long period of time 2. These factors all contribute to

the creation of a protein that can easily be incorporated into a variety of different target

molecules across a wide spectrum of biochemical identities, allowing us to easily visualize

molecules that are otherwise extremely difficult to observe.

Variations of GFP have been created to increase the range of conditions under which it is

effective, with one such variation being Superfolder GFP (sfGFP). This is generally considered

an overall more effective form of GFP; it is brighter, more tolerant to mutation, and less likely to

degrade. sfGFP is able to bind to misfolded proteins and is less likely to dimerize compared to

its wild type counterpart, and these mutations make it preferred specifically for fusing to other

proteins. We were interested in characterizing the protein’s structure and quantifying some of

the properties of sfGFP to get a better understanding of the protein’s stability. We believed that

this was a necessary step before potentially going on to use the protein in future experiments,

as by understanding the protein’s character, we could make sure to employ it when and where it

will be most effective.

Figure 1: Tertiary Structure Of GFP

Secondary structures have been colored separately to differentiate; beta sheets are colored

magenta and are in mixed parallel and antiparallel arrangement, central alpha helix colored teal,

loops are colored tan, and chromophore is colored indigo

Methods

To grow our GFP colonies we transformed 1 uL of Kanamycin resistant sfGFP-containing

plasmid solution into a 25 uL solution of BL21 (D3) E. coli cells by icing our solution for 30 mins,

and then heat-shocking by quickly pacing the solution in a heating bath for 10 seconds before

immediately removing and icing the solution for 5 mins. This procedure creates small pores in

the E. coli cell membranes for the plasmids to enter. We then added 950 uL of LB media to both

reaction solutions and incubated while shaking. After incubating, we plated 100 uL of our

reaction solutions onto LB+kanamycin plates, centrifuged the remaining experimental solution to

pellet the cells, removed any media, and plated 150 uL of solution on a third kanamycin plate. It

was important to make sure that each solution was spread evenly to avoid any clumping or

uneven spacing of colonies, as this could lead to colonies that are no longer homogenous.
Once the colonies had grown, we were given a cell-culture of which we removed 4.5 uL to be a

pre-induced sample, and then centrifuged the remaining solution to pellet the cells. It was

important that the centrifuge was balanced to ensure proper separation. We then removed the

solution media and added 5 uL of 1M IPTG to the remaining pellet to activate transcription of the

GFP encoded in the plasmid. While shaking, we took SDS-PAGE gel samples at 30 mins, 1

hour, and directly before leaving the lab —repeating the same process for each sample.

To isolate the sfGFP plasmids, we used a cultured cell-pellet from a large-scale expression to

which we added 3 ml of lysis buffer, resuspended the pellet, and then froze in dry ice for 3

minutes. We thawed the solution and then repeated the freezing and thawing procedure a

second time to create our final lysate, which we then centrifuged to separate the green

supernatant and cell-pellet. We removed any precipitate and collected 1mL of supernatant, 10uL

of which we separated to be used in our gel. To run an affinity-chromatography protocol we

added the remaining lysate to a 50% Ni bead/buffer solution and rotated to separate the green

supernatant and pellet, removing the supernatant to be used as a 4.5 uL SDS-gel sample. We

then added 1mL of wash buffer 3 to the pellet solution and incubated in the rotator to create

another 4.5 uL supernatant gel sample. We repeated this process once more using 1mL of

elution buffer 3 to get a final 4.5 supernatant gel sample

To test the purity of our collected proteins, we ran an SDS-PAGE gel for 30 mins at 200V. We

used 2X Laemmli gel loading dye containing beta-mercaptoethanol (BME)3, vortexing, and

heating to completely lyse the cells in our culture and fraction samples and denature any

proteins. We loaded 9 uL of each of our prepared samples into the SDS-PAGE gel box in the

following arrangement starting from lane 1 to lane 12: preinduction, 30 mins post induction, 1

hour, post induction, 4 hours post induction, empty, protein standards ladder, empty, crude

lysate, supernatant sample, wash sample, eluted sample, and empty. We took great care in
making sure every lane was filled neatly to reduce spilling over of samples into adjacent lanes,

as well as making sure there were no gaps in the setup of our gel box. We stained our gel using

Coomassie stain and visualized the results using the Bio-Rad ChemiDoc MP Imaging System.

For our Urea Denaturation, we prepared 11 samples of 24 uL unfolded sfGFP solution mixed

with 12 uL buffer solution and varying concentrations of 8M urea and water solution to achieve

final urea concentrations ranging from 0.5 to 4.5M. We added these samples to a 96-well plate

and measured fluorescent counts of the different samples by exciting at 485 nm using a BioTek

Synergy HTX plate reader and monitoring emissions at 528 nm. For our thermal denaturation,

we eluted our GFP into a buffer solution containing imidazole3. We then moved our GFP into a

second buffer solution using dialysis and then diluted our solution in a more concentrated

version of the same buffer. This step was necessary to remove the imidazole already present in

the GFP solution so that it would not interfere with the absorbance of 280 nm light by GFP. We

used a Cary 100 UV-Visible spectrophotometer to record absorbance at 280 nm every 2

seconds as temperature was increased by 1 degree between 25 and 80 C.

Results, Discussion, and Conclusion

Figure 2: GFP Plasmid Containing E. Coli Colonies Grown on Kanamycin Plates

(A) control plate shows displays no colony growth (B) 100 uL sample reaction plate displays

growth of few colonies © 150uL concentrated reaction plate displays growth of many colonies

Based on the colony data we can observe from the three different kanamycin platings, we can

conclude that the sfGFP DNA plasmid transformation procedure was successful. We can see

from Figure 2 that there were no colonies grown on our control plate, some colonies grown on

our 100 uL induced reaction plate, and an abundance of colonies grown on our 150 uL

concentrated solution plate. These findings were all in line with our predictions for the different

samples: the control would have no sfGFP containing plasmids with Kanamycin resistance and

would therefore produce no colonies, and the two experimental samples containing the plasmid

solution would produce colonies, with the more concentrated solution yielding a greater number.

We can also observe that the spreading was generally even, with a limited number of

connected, heterogeneous colonies formed on the experimental plates.

Figure 3: Stained SDS-PAGE of Purified sfGFP

Resulting SDS-PAGE gel treated with Coomassie stain after processing by Bio-Rad ChemiDoc

MP Imaging System; samples from left to right: time 0 mins post-induction sample, time 30 mins
post-induction, time 60 mins post-induction sample, time 240 mins post-induction, empty, ladder,

empty, crude lysate, binding supernatant, wash, first protein elution, and second protein elution

Examining our completed SDS-PAGE, we can see that the results for the different induction time

samples indicate that there was a general increase in the amount of sfGFP present as time

post-induction increased. This is visualized by the increased darkening and thickening of the

band at around 26 kD as time post-induction increased, and this also indicates that the

experimental weight of the protein is close to the literature weight of 29.7 kD. These results are

in line with our reasoning, as we accept that at longer periods post IPTG induction, there would

be more time for sfGFP expression to increase. For our crude lysate and binding supernatant

samples we can see that there was a lot of sfGFP present, indicating that the lysing procedure

was successful in removing sfGFP from the host cells, but that our binding process was not so

successful, as presence of sfGFP in the supernatant indicates that the protein did not bind well

the to the Ni beads that made up the cell pellet. This is directly associated with the lighter bands

of the remaining wash and elution sample lanes, as the reduced binding led to less protein

available to be washed and eluted. We observed this in lab also, as the supernatant of the first

elution sample was clear as opposed to green, indicating a lack of green fluorescent protein,

however the supernatant of the second elution sample was green-er than the first, giving us

hope that more GFP had been removed from the Ni beads at that point. We can assess the

purity of our samples by examining how neatly the samples are situated in their respective

lanes, and by looking at Figure 3 we can see that generally the bands for each of our samples

are relatively neat, raising our confidence that the sfGFP that we were able to collect throughout

the purification process is indeed a pure sample. We can clearly see that lane 4 does not follow

this trend, and since it is an isolated non-neat band we believe it is the result of a more isolated

pipetting error while loading the gel as opposed to a more systematic issue affecting our product

on a larger scale. Ultimately, we believe that our SDS-PAGE and all the above mentioned
results reflect that we were indeed successful in our goal of growing and purifying sfGFP, and

that while we experienced a few minor missteps such as during the affinity chromatography

protocol, generally we successfully carried out the different weekly steps to create and collect a

pure sample of sfGFP.

Figure 4: Urea Denaturation Results

(A) Graphing Fluorescent Counts monitored by BioTek Synergy HTX plate reader at each

experimental urea concentration (B) Graphing extrapolated Delta G values associated with

refolding of protein at each experimental urea concentration

To determine the stability of our sfGFP product, we can begin by examining the results from the

urea denaturation experiment. By looking at the sfGFP Urea Denaturation graph, we can see

that between 0M and 2M, the graph was fairly flat but that around 2.25M, the fluorescent counts

emitted from our samples began to drop. Since sfGFP can only fluoresce when in its tertiary,

folded state, lowering fluorescent counts indicate that as urea increased the chemical

denaturant was successful in unfolding the protein. In this graph we can also see a slight dip in

the fluorescent counts at 1M urea, which we determined to be from a pipetting error when

loading the 96 well-plate. We estimated that the fluorescent counts for maximally folded sfGFP
would be 2600 and 10 for a completely unfolded version, and we used these estimates to

extrapolate the Delta G values associated with the refolding of our proteins at each

concentration. We can see from Figure 5B that, visually, up until a little under 3.25M

concentration of urea, our Delta G values were negative, which means that energy was being

released as the sfGFP spontaneously refolded. After this point, Delta G became positive,

meaning that it required energy for the protein to refold, making it a non spontaneous and non

preferred process. We were able to use the trendline to approximate the Delta G value when

urea concentration was 0 as well as the concentration at which the protein was 50% folded and

unfolded (Cm). The values we received based on our trendline were -14.9 kJ/mol for Delta G(0)

and 2.7M for Cm. Comparing the trendline data with the actual data points, we can see that

these values do not match up very well (as evidenced by the relatively poor R^2 value). The

actual data suggests that both values should probably be a bit higher. We believe that this was

partially due to the pipetting error at 1.5M, as well as a more general discrepancy between the

shape of the curve and the linear trendline superimposed onto it. In the future, we will

experiment with either refining our experimental procedure to eliminate these errors, as well as

researching better fit models to represent our data.

Figure 5: Thermal Denaturation Data

(A) Graphing Absorbance of 280 nm light monitored by Cary UV-Visible spectrophotometer

every 2 seconds between 298 and 348 K (B) Graphing extrapolated Delta G values associated

with refolding of protein at each experimental temperature of sfGFP solution

To round off our stability experiments, we can examine the data collected from our thermal

denaturation. As sfGFP unfolds, tyrosine residues are exposed which contribute to the

absorbance of light at 280 nm. By looking at Figure 5A, we can see quite clearly that the

structure of sfGFP remains stable in its folded state until temperatures begin to exceed 320 K,

as at this point absorbance rates increase telling us that the protein is unfolding. Using a similar

method as used in the urea denaturation, we were able to extrapolate the Delta G values

associated with the refolding of our protein at each data point. We chose to behind our graph at

315 K as this was the region in which unfolding began. We used the trendline of this graph to

estimate the temperature at which the protein was 50% folded and unfolded (Tm), and the value

we got we felt was much more representative of the actual data. The Tm value based on the

trendline was 335.0 K, which when compared to the actual data graphed, we can see it lines up

very nicely. This result gives us confidence that our thermal denaturation procedure was carried

out without any major missteps. Together, the data from the urea and thermal denaturations give

us a much clearer picture as to what the character and properties of sfGFP are. When designing

experiments for future research, we now have a better understanding of which conditions will be

best for the more efficient use of sfGFP.

 References

(1) Reid, B. G., and Flynn, G. C. (1997) Chromophore formation in green fluorescent
protein. Biochemistry 36, 6786–6791.

(2) Carolina.Biological. (2021, December 3) How green fluorescent protein is used in

research. Carolina Knowledge Center.

(3) Pollet, Rebecca; Turtle-Schmidt, Brian; etc. Biochemistry Essentials: Purification and
Characterization GFP, A Laboratory Manual to Accompany 272: Introduction to
Biochemistry; Vassar College: Poughkeepsie, NY, 2023; p. 15, 19, 24