Today Nahir and Oleg gave presentations on their biochemical projects involving AdoMetDC, which is a pro-enzyme for which T. brucei needs in order to synthesize necessary amino acids. The sequence of this pro-protein at the N terminushas been concerved across several species excluding mammalian species including humans. This makes it a viable protein for drug targeting. Nahir's project involves creating a better way to produce more of this target protein in order to purify the protein in it's entirity for crystallography purposes. It seems her first problem was mainly getting the N-His tag to come off the protein without disrupting it's enzymatic activity--you need to be able to prove that and therefore you should be able to first cleve the N-His tag. She had problems doing that. So she cloned the gene into a vector in a new way that included a novel product known as th e SUMO fusion sequence. This sequence assists the protein in it's cloning and facilitates folding, therefore increasing the amount of viable protein. She first designed her primers and also designed primers to cleave portions of the N terminus off the gene, later testing for enzymatic activity. How to test for enyzmatic activity and not just folding? Immuno-assay. or is it co-immuno precipitation assay?
So to ensure the gene was in the new vector she ran a gel. Showing the reaction inserted. She may also may have had to do some mini preps. She then digested the vector to show that she could isolate the gene and put it back inside. Isolating the gene meant she needed to sequence it.
confirming the correct gene she then began the process of large scale bacterial cultures. She'd lyse the cells and then purify the protein with the Ni2+. Through a 2 step process of running the solutions through the column on a gradient, she was able to isolate the protein without the His-tag. She would run gels using affinity chromatography which elutes the proteins based on size. In some mutants with a more upstream N-terminus deletion she lost the pro-enzyme associated with AdoMet. Ideally you want a clean protein for crystallization purposes.
Oleg was having trouble eluting enough of his protein. Seemed that in purification processes he would lose more than half of his protein. It seemed that there was too much protein and the column was too old to handle becoming overloaded. It was recommended to run more batches but diluted and with a better column.
