Conceptualization
Our validation assays must use a more direct proxy to validate the performance of the tag. Thus, we elected to do densitometric analysis of the relative protein amounts based on literature review.
Monoclonal variants that have undergone selection would have been cloned into plasmids with pre-selected host proteins. This would be done along the original variant. These plasmids would then be transformed into BL21 E. coli. These are then cultured and induced to produce these protein fusions, alongside untagged hosts.
The three host proteins chosen were based on the relative ease of performing the functional assay, size, and degree of aggregation.
Solubility Assay
The solubility assay was originally intended to load whole lysate, soluble fraction of lysate and insoluble fraction of lysate onto SDS-Page gels and perform densitometric analysis to quantify relative amounts of proteins in each band.
The signal of the insoluble to soluble protein would be compared against the different tags to see improvements in solubility. The comparison of the whole lysate against other whole lysates would have been used to see the impact on expression. Together, these data would be able to conclude the degree of impact the tags had on expression and the degree of rescue.
Lysis
The lysate had to be produced via a method that did not disturb the inclusion bodies formed. This is to generate an insoluble fraction for loading. Chemical lysis was conducted by resuspension of cell pellet in 1 mL of lysis buffer (20 mM Na3PO4, 500 mM NaCl, 20mM imidazole, 0.2 mg/mL Lysozyme, 20 μg/mL DNAse A, 1 mM MgCl2, 1mM PMSF). The lysate is then lysed via mechanical lysis through three freeze-thaw cycles and three bead disruption cycles.
SDS-Page Gel
A 12% Tris-Tricine gel was used to resolve all samples due to ability to resolve a range of protein sizes that contains all proteins of interests.
Pilot Experiment 1
The lysate of GB1-isPETase fusion was used. 50 uL of the lysate was centrifuged. The protein concentration of the lysate was measured in order to determine ideal dilution for sharp bands. This led to three dilutions being tested: x1, x10, x100. 5 uL of the whole lysate was used as the Total fraction (T). 5 uL of the whole lysate was spun down. The supernatant was transferred as the Soluble fraction (S) and the pellet as the Insoluble fraction (I).
The samples were then prepared with LDS Sample Buffer x1, 50 mM DTT and incubate at room temperature for 30 minutes before loading.
The results of the pilot study showed that at all dilutions, sharp bands could be seen, however, no significant differences in terms of band composition were seen across the three fractions. Slight differences in the brightness of the tag were seen. It was suspected that some of the soluble fraction may have remained in the insoluble fraction, which will be resolved if washed.
Experiment 1
The experiment was repeated with all strains with one modification. The pellet was washed with 5 uL of lysis buffer twice with the washings loaded. This is to ensure no soluble fraction contaminates the insoluble fraction.
The results of the experiment were inconclusive due to the incompatibility of our protein ladder. However, a clearer difference between the fractions is seen. The washings did not contain many bands which indicates that the clutter of proteins in the insoluble fraction are the E coli native aggregation prone proteins.
No discernable differences between different strains led us to believe that our proteins may have failed to be expressed due to various factors.
Experiment 2
The experiment was repeated using a purified soluble fraction. Insufficient time and technical abilities prevented the purification of a solubilized insoluble fraction.
The purification proved whether our protein of interest is present. The purified protein and its respective fractions were loaded. Two of six fusions were identified. Densitometric analysis can be performed on the identified band and normalized with a common housekeeping protein. This can now be compared to measure the relative change in protein amounts, which is indicative of solubility improvements, whereas the change in the whole lysate is indicative of changes in expression level, thus fulfilling criteria for validation.
Further work must be done to complete the assay.