Experiments

Part IV - Determination of the best purification system

Introduction

The goal is to test and compare the Gluthation purification system and the hisTrap one to systems to see which one would be the most effective for our box. To do so, we have purified GST-10XHIS proteins with both of the purification system. We have determined the GST enzymatic activity before and after the purifications to obtain the purification yields. Finally, we have done SDS-PAGE chromatographies to semi-quantify the proteins and compare the results with those obtained with the enzymatic activity.

Results

We cultured DH5α containing plasmid pet21 GST-10XHIS. We measured the OD every hour in order to follow the turbidity and thus determine the time corresponding to the exponential phase of growth(fig.11).

Figure.11: Growth curve of the DH5α containing the PET 21 GST-10HIS
All the points data come from a mean done by data collected from 5 cultures. Cultures were incubated in LB at37°C. The DO(600nm) was calculated every hour.

Analysis of GST enzymatic activity:

We calculated the GST activity of the sample taken before and after the purification. We took 5µL of the aliquotes to do the reaction. We then calculated the total reaction for the all volume before and after lysis that can be found in table.1. The table.2 presents the the activities and purification yields obtained for both of the purification.
For the HisTrap column, we observe a purification yield equal to 72.1 %.
For the Gluthation Agarose Resin we observe a purification yield equal to 32%.

Fraction	Enzymatic activity From 5 µL (Δabs/min)	Total enzymatic activity (Δabs/min)	Purification Yield
F1 HisTrap column	0.13	208	72.1%
F2 HisTrap column	0.15	150
F1 Gluthation Agarose Resin	0.75	120	32%
F2 Gluthation Agarose Resin	0.77	38.5

Table.2 GST Enzymatic activity obtained and purification yield

Analysis of SDS-PAGE gel:

After bacterial lysis, we prepared aliquots from the solutions before and after purification and we performed SDS-PAGE for both types of purification. This is done in order to check the presence of GST-10XHIS and to compare these results with those obtained with the enzymatic analysis.

12% polyacrylamide gel. 10 µL deposited with Laemli 1X for each well. M: Kb Ladder. 1: eluted and purified proteins using a Gluthation-Agarose column. 2: expressed proteins before purification.

Figure.13: SDS-PAGE analyse of purification of GST-10his proteins using a HisTrap column

Before purification, mutliple bands can be seen corresponding to bacterial proteins. However, an intense band at about 27 kDa, which correspond to the GST-10His is observed and suggest a good expression of the protein(fig.12.2). After purification with Gluthation Agarose Resin, we can only see one band corresponding to the GST-10His but smaller than the one seen before purification(fig12.1).

SDS-PAGE of HisTrap column purification : Similarly to what was observed in the previous SDSPAGE, multiple bands and one more intense can be seen at about 27kDa corresponding to the GST-10His, before purification(fig13.2). After purification, only one the GST-10His band is observed at 27kDa. The band looks as intense as the one before purification(fig13.1).

Comparing the results with the SDS-PAGE and the GST enzymatic activity, we can say that the purification yields are consistent with the intensity of the bands observed with the SDS-PAGE. Indeed, with the Gluthation-Agarose column, we calculated a purification yield of 32% which is consistent with the diminution of the band size after purification. Likewise, the purification yield of 73% observed with the HisTrap column is consistent with the size of the band. However, the size of the band after HisTrap purification seems to be the as the one before purifcation so the purification yield should be closer to 100%. We have probably lost GST enzymatic activity during the experiments.

To conclude, the HisTrap column seems to be more efficient at purifing the GST-10His protein than the GST-Trap one. Yet, we should not forget that the HisTrap purification System require buffer containing imidazole. Imidazole is irritating and can cause allergies, so we will have to be even more careful when choosing our following Sephadex exclusion chromaztography to exclude small undesired composants. We should maybe try other types of column like the one capable of binding the SUMO protein for example. For the box modelisation, we still decided to use the data obtained with the HisTrap column.