From the protein was not folded properly. Similarly,

From the SDS PAGE, the expression of protein was observed in induced, insoluble fraction, soluble fraction and eluate fraction at molecular weight ~50kD. The bands expressed between 37kD and 50kD indicates for DHFR. The enzymatic activity of DHFR was calculated as 2.8× 10-7mol L-1min-1 using Beer’s Law. After SDS PAGE analysis, the band was observed in induced sample (lane 2) was due to presence of GST-DHFR-His5. Due to absence of GST-DHFR-His in an uninduced sample no band was observed in lane 1 in SDS PAGE. The band observed in an insoluble fraction explains the GST -DHFR-His was aggregated or the protein was not folded properly. Similarly, being insoluble it settles down with the debris including cell wall, cell membrane5. The band observed in soluble fraction lane determined the lysis occurred. GST in DHFR increases the solubility of the protein. In the presence of larger strongly expressed band with molecular weight approximately 12kD, represents the lysosome that is involved in lysis of the cell.Theoretically, due to presence of lesser amount of GST-DHFR-His in the flow through, not even a single band should be expressed. But in this case, the band was observed in flow through. This result is might be if a certain amount of DHFR protein was not able to bind with the nickel in IMAC resin in the spin column. GST-DHFR-His was in higher amount in eluate fraction, because imidazole competes with six histidine’s of GST-DHFR-His and breaks the bond between histidine and Ni-sites. This might be the reason why the band was observed in eluate fraction. In the experiment the band observed in desalted eluate fraction was not strongly expressed. Originally, the band in desalted eluate fraction should be of same strength because this salting process removes the imidazole, but it does not affect the eluate fraction added in desalted fraction. As depicted in figures 2A and 2B, the enzymatic activity of DHFR is highly effective.While performing an experiment, the results did have some deviations. These deviations might occur due to some technical errors like expiration of gel, overloading of sample, cross contamination in SDS PAGE. Despite some technical error, overall experiment performed was successful.