Registration for a live webinar on 'Gamma-delta T cells for immunotherapy of cancer' is now open.See webinar details
Designing proteins with life sustaining activities 2
Published on April 3, 2017 24 min
A selection of talks on Biochemistry
The ERK1/2 MAPK cascade
- Prof. Melanie H. Cobb
- University of Texas Southwestern Medical Center at Dallas, USA
Amino acid conjugation: mechanism and enzymology
- Dr. Kathleen Knights
- Flinders University, Australia
So the big question is, how do the de novo proteins function? Two alternatives. It would seem logical that the de novo protein catalyzes the same reaction as the deleted enzyme. That's seems totally reasonable, right? However, that's not necessarily the case. It might also perform the rescue by somehow increasing the expression or enhancing activity of some endogenous natural E. coli protein. Hence, we have to consider both possibilities, the first simple one and the second more complicated one. So which is it? And the answer is, yes, it's both. We have examples of both cases. Let me go through the second one first and then I'll go through the top one afterwards. So enhancing the activity or expression of an endogenous E. coli protein.
On this next slide, I'm introducing you to SynSerB, and so that indicates the synthetic protein Syn that rescues the deletion of SerB, okay, and SerB is the gene that codes for phosphoserine phosphatase, that's essential in the biosynthesis of the amino acid serine, and the key reaction is shown on the top right. It's a phosphatase that removes out phosphate group from phosphoserine to yield the essential amino acids serine. If that gene encoding that enzyme is deleted, then the cells cannot grow on minimal medium. As you see in the panel on the bottom left, if we grow cells in minimal medium, delta SerB cells, these deletion cells, in minimal media, if we put in the control gene encoding the control protein LacZ, nobody grows, dead, baseline, flat line. However, if we put in the wild type, the natural SerB, things grow rather quickly as you see in blue. Alternatively, if we put our synthetic protein in, or more specifically, a synthetic gene encoding our synthetic protein, we get growth, not surprisingly, it's not as good as natural wild type, but yes, surprisingly, it actually works. This novel protein sustains life. So how does it work? We purify the protein and we see that in vitro the purified synthetic protein does not catalyze the reaction showed on the top right. Perhaps there is something in the cell lysates necessary. Nope, the cell lysates don't do it either. So we're forced to the conclusion shown in purple here that the rescue mechanism is not catalytic but somehow regulatory. But we don't know what it is. We really have no idea where to look. And so we did a rather involved experiment here called RNA-seq, RNA sequencing, and we asked which genes in E. coli, which RNA transcripts are enhanced, are turned up by the presence of our synthetic protein, our artificial protein SynSerB.