Flappase Characterization
Expression Optimization
The 10X His tagged Flappase expression vector was transformed in BL21 DE3 competent cells and verified via colony PCR. This composite part included the inducible promoter (part BBa_I719005). We chose to characterize the expression of our Flappase part in the construct with this promoter with a series of small-scale experiments to determine the optimal conditions for the highest protein expression.
Figure 1: Verification of the Flappase expression construct (credit, Team SUNY_Oneonta, 2020). The Flappase construct was designed and ligated into pSB1C3. Ligations were then transformed into E. coli BL21 cells. (A) Colonies were screened using colony PCR to identify clones containing the insert. (B) Minipreps and restriction digestions were used to confirm insert.
In order to test the expression of the Flappase protein, we set out to test various concentrations of IPTG to induce production and determine the optimal length of incubation prior to harvesting the cells. We transformed the 10X His tagged Flappase part (BBa_K3389003) into BL21 DE3 competent cells using our transformation protocol. We confirmed a successful transformation via colony PCR and restriction digest (see Team SUNY_Oneonta 2020 wiki).
Figure 2 summarizes the small-scale expression experiments conducted to determine optimal conditions. In these experiments, each test tube contained 3mL of the transformed Flappase construct as a liquid culture with 25 ug/mL chloramphenicol. Cultures were grown until they obtained OD600=0.600-0.800, then were induced using IPTG. The following concentrations were tested: 0mM (no IPTG, this served as our negative control), 0.5mM IPTG, and 1mM. We had three test tubes set up for each concentration, each test tube served as a time trial for our harvesting time. Nine test tubes were set up in total, 1mL was harvested from each tube after three hours, four hours and overnight.
Figure 2: Determination of optimal growth conditions. Small scale cultures were grown to determine conditions for maximum Flappase expression. Each sample set was harvested at a different time post-induction with IPTG. Sample set A, was harvested after 3 hours, sample set B after 4 hours, and sample set C was left to incubate overnight.
Cells were harvested using high speed centrifugation and washed with PBS buffer twice in order to ensure most of the cell debris was discarded. Then, we proceeded to lyse the cells via sonication. We conducted another set of experiments to ensure complete lysis. The cells were lysed via sonication for a total of 1 min, 3 mins, 5 mins, 7 mins and 10 mins. After sonication, the samples were subjected to SDS-PAGE and the gel was stained with Coomassie (Figure 3). The one-minute sonication sample yielded very strong bands at ~42kD, which is the size of the desired Flappase construct. Not only are the bands the correct size, but they are darkest in color which means there is a high protein concentration present. The samples that were sonicated longer also show strong bands at the correct size. This tells us that only 1 minute of sonication was required to achieve complete lysis. We opted for the 3-minute sonication time to be sure that cells would be lysed, regardless of the viscosity of the samples.
Figure 3: Optimization of cell lysis by sonication. SDS-PAGE gel showing the results off lysing cells using 1, 3, 5, 7, and 10 minutes of sonication time. All samples show a band at 42kD, the size of Flappase. No benefit was seen with respect to total protein yield beyond one minute.
The induction time trial samples were imaged on an SDS-PAGE gel in order to visualize which set of conditions is ideal for the highest yield of the Flappase protein. The optimal conditions for expression and induction resulted in 1mM IPTG harvested three hours post induction.
The western blot indicates that our inducible promoter in the Flappase construct is functional, but it is very weak. The faint markings in each lane show that the promoter is producing the protein at all concentration levels. Only the overnight samples showed significant dose-dependent induction of Flappase. At the 1mM IPTG induction level with overnight incubation, we see the darkest line in the Western blot, and this shows that this sample contained a much higher concentration of the protein of interest.
Figure 4: Western blot of induction time trials. All samples were lysed for 3 minutes; total protein loaded was the same in each lane. The His-tagged Flappase protein was visualized with an anti-His antibody. The yield of Flappase is much higher when induced with 1mM IPTG and left to incubate overnight.
Purification Optimization
We chose to use Immobilized Metal Affinity Chromatography (IMAC) to purify the His-tagged recombinant Flappase. The 10X His-tag in the Flappase construct (part BBa_K3389003) makes IMAC an easy way to purify our protein for use in the Flappase Assay, as the His-tagged proteins are attracted to the nickel of the IMAC column used. Flappase was purified from cell-free extracts using a Bio-Rad NGC medium pressure liquid chromatography system fitted with a 5mL Nuvia IMAC Ni-charged column. Throughout the optimization process, we adjusted the chromatography system based on our results. Figure F-05 describes several schemes that were used in the optimization process.
a.) IMAC Scheme 1– Linear Gradient Elution
Buffer A – 300mM Sodium Chloride, 50 mM Sodium Phosphate
Buffer B – 300mM Sodium Chloride, 50mM Sodium Phosphate, 500 mM Imidazole
Equilibration
Flow rate: 1.00mL/min
Isocratic - 100% Buffer A, 0% Buffer B, 5 column volumes (CV)
Sample Application
Flow Rate: 1.00mL/min
Volume: 1mL
Fraction size: 0.5mL
Column Wash
Flow Rate: 1.00mL/min
Volume: 3 CV (1 CV = 5mL)
Isocratic – 99% Buffer A, 1% Buffer B
Fraction size: 1mL
Elution
Flow rate: 1mL/min
Fraction size: 0.5 mL
Gradient Elution: 1% B to 100% B
b.) IMAC Scheme 2- Isocratic Elution
Equilibration, Sample Application, and Column Wash identical to Scheme 1. The Elution phase was altered as follows:
Elution
Flow rate: 1mL/min
Fraction size: 0.5 mL
Isocratic elution: 100% B
Figure 5: Optimization of Flappase purification by IMAC. (a.) Initial IMAC protocol used for purification¬-This scheme was not very useful, as the gradient elution became difficult to analyze post-purification. We opted to change the elution for following protocols to only contain isocratic elution. (b.) Purification scheme optimization process. Due to the lack of success of Scheme 1, we altered the settings of the elution phase. Isocratic elution at 100% B was tested.
Elution fractions were chosen for analysis based on the significance of the peaks in the associated chromatogram (absorbance monitored at 280nm). Each peak indicates the presence of eluted protein. We selected peaks from the wash phase and elution phase. Further analysis via SDS-PAGE was done to identify fractions containing proteins of the desired molecular weight.
Figure 6: IMAC chromatogram, 500mM imidazole elution. The following fractions showing significant peaks were selected for analysis: 2, 10-14.
Figure 7: SDS-PAGE analysis of elution fractions. CFE, cell free extract; WF2, wash fraction #2 (20mM imidazole); EF10-14, elution fractions 10-14. Fractions 10-14 were eluted with 500mM imidazole.
From the IMAC analysis and the SDS-PAGE gel we were able to obtain for these samples, we noticed that our schemes required more changes. The SDS-PAGE indicates that the suspected protein of interest is being released during the wash phase of the purification scheme, due to the presence of a band at ~42kD in the WF2 (Wash Fraction #2). Elution fractions EF12-13 (Elution Fraction 12-13) also have a visible band at 42kD, along contaminants at other molecular weights. The precise location of our His-tagged protein will need to be confirmed via Western blot. Unfortunately, due to time constraints we were not able to complete this or to run another scheme with a different wash buffer setting.