Find more mlrA gene
In order to express the protein, our team combined the gene of interest with multiple vectors. After running the gel electrophoresis, we found the protein was failed to express in the clear supernatant extract. Last year, SBS _SH _112144 team extracted mlrA gene from Sphingopyxis sp. C-1, an alkalitolerant microcystin-degrading bacterium. This year, the same experiment was also performed on different genes of mlrA selected from different species, ruling out the possibilities of choosing the wrong target genes. The mlrA gene we used in the experiment were extracted from species Novosphingobium sp.THN1 and Shingomonas sp. separately, and were tagged as mlrA2 and mlrA3 for experiment purposes. The result was still negative for protein expression in the supernatant extract. We blast the sequence and search for more mlrA gene from different species to find a best activity expression in E. coli.
Then we selected a dozen of candidate gene.
Table.1 the two rows highlighted in yellow were the mlrA genes used in our team experiment
2.Predict the structure of mlrA
Through multiple gel electrophoresis, our team discovered a pattern: the protein is always expressed in the sediments. We did some research online and found that, according to Paola Marani’s paper on New Escherichia coli outer membrane proteins, integral membrane proteins cosediment with the outer membrane. Thus, we predicted that mlrA is a membrane protein, which will subside in the sediment along with other lysed cell debris. The sequence of the MlrA gene was analyzed by the website, raptor x prediction. According to Raptor X Predicton, the summary prediction results shows the secondary structure of mlrA2 contains 63% helix, 0% strand and 36% coils.
Figure.1 Summary Prediction Result of MlrA2_SS by Raptor X
Similarly, the secondary structure of mlrA3 contains 64% helix,0% strand and 35% coils.
The result confirmed that the secondary structure of this protein is a helix structure within bilayer membrane. According to Francesca M Marassi’s paper, NMR structural studies of proteins, three different polypeptides in oriented bilayers all have been shown to have predominantly helical secondary structure by using multidimensional solution NMR spectroscopy in micelle samples. The analysis of the secondary structure of the target protein appeared to be in accordance with the description in Marassi’s paper. Tertiary structure of the this MlrA gene also accorded with the structure of membrane protein. Therefore, the data supports our prediction that MlrA is a membrane protein.