Figure 1:SDS-PAGE of the expression proteins. Lane1: Ladder; Lane 2 & 3: Before induction; Lane 4 & 5: After induction.
ION exchange chromatography:
Ion exchange chromatography was done to purify the protein of interest. The protein of interest was E2 which was found to be expressed in the bacterial cells (E.coli DH5α) using the plasmid pRSF_ T6 Ube2t. The protein of interest cloned was MCS1 which is His6- Ube2t (His tagged).
On separation of protein fractionation, the supernatant was allowed for ion exchange to elute the protein of interest. The protein of interest was found to be approximately around 24kDa. As shown in the figure 2, the protein of interest was found at a peak of molecular weight 24kDa between the 35-40ml of elute volume. The sample component was collected and later used for the SDS PAGE analysis for validation.
Figure 2: Ion exchange chromatogram showing the peak value of the desired protein. The protein of interest peak was shown to be at 24kDa. The volume of the elute between 35-40ml.
The samples run on SDS PAGE showed that the fractions are pure in nature
The protein band of interest was found to be approximately between 25 and 23kDa (data not shown) which was correlating to the hypothetical calculated value (24638.0410) from the online calculator. The protein bands confirm of the presence of the desired protein. All the samples were pooled and used for the Ubiquitination assay.
The E2 conjugating enzyme tested in the reaction catalysed the substrate ubiquitylation assay. From the data it seems likely that E2 conjugation with the substrate was possible at 30min and thereafter. In lane 2, the substrate was free in nature, whereas in lane 4 the substrate conjugation was seen clearly. This clearly indicates the role of E2 in conjugation. As from the figure 3, the conjugation increased with the time. In lane 6 the conjugation of E2 can be clearly depicted.
Figure 3:SDS-PAGE showing Ube2t Ubiquitination. Lane 1: Protein ladder; Lane 2: 0 minutes samples with ATP; Lane 3: 0 minutes samples without ATP; Lane 4: 30 minutes samples with ATP; Lane 5: 30 minutes samples without ATP; Lane 6: 60 minutes samples with ATP
From the figure 4, it is clearly understood that the E2 conjugation was decreased as the time increased. In lane 1, the conjugated sample was found to be deconjugated. In lane 3 and 4 the deconjugated substrate can be seen away from the E2. This clearly depicts of the role of E2 in conjugation.
In the figure 4, E2 protein was observed at about 24kDa. Once DUB was added initially, the deconjugation of the protein can be seen clearly. In lane 3 and 4, the deconjugated substrate was found to be separated at about 50kDa.
Figure 4:SDS-PAGE of Deubiquitination. Lane M: protein ladder; Lane 1: 0 minutes with DUB; Lane 2: 30 minutes with DUB; Lane 3: 30 minutes with DUB. The samples were run on 10% gel.
Ubiquitination is a post-translational modification which is reversible in nature and extensively used during regulation. The attachment of ubiquitin to some of the cellular proteins influences many such pathways and inturn disrupts the protein localization and proteosome degradation. This regulation is linked to many diseases in humans like cancer, neuronal dysfunction and muscle wasting diseases. The E2 conjugating enzyme can either directly bind to E3 or directly transfer the ubiquitin to the target protein. In order to carry this function, E2 binds to E1 and activated ubiquitin. Humans possess about 35 different types of E2 enzymes which are highly characterised and highly conserved in nature. The results obtained were found to be successful. The proteins isolated were quantified using the UV spectrophotometry and was found to be pure. The concentration of the protein sample was found to be promising for the filtration techniques. From the figure 3, it was observed that the band was our protein of interest with an approximate molecular weight of 23- 24kDa. The sample obtained was confirmed of the E2. This sample was further purified on gel exclusion chromatography. The elutes were pooled for the total fraction and used for the ubiquitination assay and deubiquitination assay.
The main approach of the study was to confirm of the assertive role of E2 in autoubiquitination. As the experiment was done in the absence of E3, the role of E2 was possible defined and understood. The data clearly shows the role of the E2 in directing the ubiquitination. From the results, it was understood that the E2 aids in ubiquitination. And from the present study our findings confirm the possible individual role of E2 in conjugation. As E2 is thought to act lonely or in conjunction with E1, the activity was mainly to confirm the role of E2. Hence as the study was without E2 and the results also shows of its nature. Further experiments need to be planned on ELISA which shows specific activity. Antibodies can be designed for the E2 and E3 and can be carried using the sandwich mode of ELISA. Further it can also be confirmed on the knock out cell lines. Using RNAi technology, specific E3 knockouts can be created and used for the experimentation. Studies on knockouts confirm the findings not only at biochemical level but also at the molecular level. On summing up, the E2 protein can be studied in detail about its mechanism and its action. This can unravel many questions of genetic disorders in humans. This peculiar feature of E2 conjugating enzyme to complete the cascade in the absence of E3 would surely be of interest for the scientific field. Moreover, we can expect a better understanding of the prognosis of the diseases and in developing diagnostic tools.
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