Team:Tec-Monterrey/allergen/results

Shuttle Expression Sequence

For P.pastorias, the Shuttle Expression Sequence was designed to remove an intron that functions as a promoted in E.coli (B.P.S), this way, the same protein can be produced in P.pastoris and E.coli from the same DNA sequence. Expression of Der f 2 in E.coli showed that metal affinity proteins from the innate proteomo of the bacteria generated considerable bakcground noise. Additionally, deficient affinity of the Histidine tag was detected; to deal with this issue, denating conditions (8M Urea) were assessed to try and expose possible unexposed Histidine tags. Differential elution conditions are being evaluated to reduce affinity of contaminant proteins and prevent the loss of Der f 2.

Tricine SDS.PAGE (16%) under reducing conditions.
Lane 1: P.pastoris supernantant pot -IMAC 16.6KDa Der f 2 (SES)
Lane 2 and Lnae 3 : MOlecular weight marker.
Lane 4: E.coli (BL21 STAR JM109 Rosetta-gami) denatured (Urea BM ) and purified by IMAC 16.6 KDa Der f 2 (SES)

P.pastoris Protocol and E.coli Protocol

Production of scFv in Pichia pastoris

Tricine SDS-PAGE (12%)under reducing conditions. Lane 1: P. pastoris supernatant 27.4 KDa Anti-His scFv
Lane 2: P.pastoris supernatant 53 KDa Anti-IgE fused yeGFP
Lane 3: Molecular weight marker
Lane 4: P. pastoris Anti His soluble & insoluble fractions
Lane 5 P.pastoris Anti IgE fused yeGFP soluble & insoluble fractions.

Anti-IgE scFv fused yeGFp (53KDa) was detected by SDS-PAGE in the supernatant fraction, indicating secretion of the protein by P.pastoris, however the anti-His tag scFv (27.4 KDa) seemed to present very low expression of the mentioned scFv (both transformants were used as a control to each other). Another band from Anti-IgE scFv fused yeGFP was also detected in the soluble fraction of the cell lysate while no presence of anti-His tag scFv was detected.

Pichia pastoris Protocol

Activity assay Anti IgE scFv and Anti His scFv

For these assays, the concentrations of the scFv's were assumed to be 0.5% of the total amount of protein in the extracts. Unwashed wells were used to measure the maximum possible signal from the GFP, while wells loaded with non binding GFP were employed for determining the efficiency of washes. ANOVA tests showed that there was no significant difference between Blanks and wells with binding GFP at any concentration in either assay. We believe this can be due to the high concentration of contaminant proteins in the extracts or further variables not considered in this assay.

ELISA test to measure the activity of our Anti-IgE scFv+ye GFP. The well coated with 100uL of concentrated human IgE ( 2ug/mL).

ELISA test to measure the activity of our Anti-His tag scFv. The assay was run two times with wells coated with 100uL of Anti-His tag scFv at two different concentrations (2ug/mL & 4 ug/mL)

GFP analysis…

To prove the activity of scFv IgE+GFP, we needed to detect both parts separately. To de GFP emission we designed the following experiment.

Using a 96-well fluorescence plate (a black one) we filled some wells with supernatant from our induced Pichia pastoris with scFv IgE+GFP, some with the intracellular extract from the same culture, and the same treatments but for our P.pastoris with scFv His. The fluorescence plate for the experiment was designed as follows:

Where GFP III, IV, V, VI are different P.pastoris strains expressing scFvIgE+GFP, Api is a P.pastoris strain expressing Api m 6, His is a strain from the same organism expressing scFv His, SN means supernatant , ICs refers to the soluble fraction of the intracellular extraction and Blank is just media without any strain.

The results were organized on the following tables, the first one comparing the results between the different GFP strains and their fluorescence in supernatant and intracellular-soluble fractions and the second one comparing one of the GFP strains to the other ones.

In average, the results seem to mean that we indeed have a fluorescent protein on the desired strains and that it does exists more in the supernatant, thanks to the signal peptide is was designed to have. But just that is not enough to approve anything, to interpret these results with more confidence we need to do further mathematical analysis.

The ANOVA test was chosen because we have al the conditions needed for its application. So two ANOVA tests were done, the first one to see if there was any difference in fluorescence between the GFP strains and/or between the fraction (supernatant/ intracellular soluble), and the second to see the same difference but between one of the GFP strains and the His/Api extractions.

Thanks to the ANOVA 1 test, now we can conclude information with certainty. The ANOVA test table above shows that our statistic Fp for the source A is greater than the ft, this means that data from the source A (fraction) is significantly different with a 95% percent if confidence. The Fp of source B being not greater than its ft, means that there is no significant difference between the fluorescence of the GFP from different strains. Finally, the AB source having a Fp less than its ft means that there is no difference in combinations of source A and B, which is not of our interest. This means, that yes, we can conclude that we have more fluorescence in the supernatant and that all of our GFP strains are expressing the same with at least 95% of confidence.

The ANOVA 2 test was designed so that it would tell us if we had any difference in fluorescence between a GFP strain and the other non-GFP strains. Using the same logic that in ANOVA 1 test and comparing the Fp’s from the ft’s, we can conclude with at least 95% of confidence that our GFP is different in fluorescence that our other strains and again we can conclude that this protein is being secreted. In short, the GFP part of our scFv IgE+GFP does work and it is being secreted successfully!