Probably all the proteins encoded by the human genome would denature and fail to function in the environment occupied by the hyperthermophilic archaeon Geogemma barossii. Clearly proteins are expected to work in the environment under which they are selected, not in some other arbitrarily thought up condition or organism with which they have nothing to do.
@Mercer What it means is, you start over until you really do have activity by changing just the things you mentionned, and we have discussed. I have to check the paper yet. But if they didn’t do those things, and just declared they had identified 5 beta lactamase catabolic antibodies capable of carrying out beta lactamase reaction _without quailification_that would be wrong.
Yes. (Think before you reply.)
@Mercer Where does this come from? I know what ATP is, and kinesin and myosin. What does it have to do with this discussion? I was not talking about Szostak’s random sequence library.
I have worked with proteins extensively, and what you describe makes no sense. No activity in the end means you did something that denatured or inhibited the protein. Since there was activity before this point you already know that it has activity. For example, I studied a metalloprotease at one point, but I didn’t know that it was a metalloprotease at the time. I added EDTA to one of my solutions, and I couldn’t figure out why I lost activity. Did this mean the protein never had function? NO!! What I did was strip out the divalent cations that it needed for function.
I don’t know of any enzyme that has function in every possible aqueous environment. In fact, one of the first things I often did was test for activity at different pH’s, different salt concentrations, in the presence or absence of detergents, and so on. I also know from experience that there are tons of proteins that are functional AND insoluble in water (e.g. many membrane proteins).
What you are describing doesn’t make sense to me. If there was beta-lactamase activity associated with that clone then there was activity. Period.
Me too, and I agree completely.
@T_aquaticus Sorry I was unclear. If you had already gotten to the point where you could add EDTA then you were already in aqueous solution, you already had your protein. I meant if you never made it to the aqueous phase–for example, if your protein was only soluble in acid.
It’s a good thing Ann doesn’t work with RNA.
Acid is aqueous, so I’m not sure what you are getting at. Pepsin has a pH optimum under 2.0, so is it not a functional protein? If I am reading it correctly, beta-lactamase activity was tested for at a pH of 7.4 in wells with bound phage. So there was activity at near neutral pH.
Note, the discussion on catalytic antibodies is important, and should be split soon by @moderators into its own thread.
My favorite grad-school applicant interview question to assess lab savvy is, "You’ve got two tubes. One has DNA, the other RNA. You accidentally spill both on the bench top. Which one(s) would you try to salvage, and why?
@Agauger, this seems to be an important point. I have not read the paper closely yet (sorry), but there seems to be a disagreement on the material facts here that should be fairly easy to resolve. You argued that these clones did not have activity in solution after being separated from the phage proteins. I’m not sure why that matters, but @art and @T_aquaticus is saying that they did have activity in solution. Do you dispute this still?
For what it is worth, there is a soluble antibody produced by living cells that has beta-lactamase activity:
It would be so much easier to have a civil discussion without gamesmanship and point scoring and accusations. I already apologized for the error in your paper as I recall. That was an unpleasant experience, mainly because no one would give a straight answer. And I was badgered and sneered at and mocked,
So bringing up here is not exactly helpful.
What is your purpose? Is it to humiliate me?
Or is it to persuade me?
You are unlikely to get both.
I’m bringing it up because you’re making a similar tactical mistake today. You’re trying to push against a mountain of evidence by holding up a few words.
This seems to be an even more important point, and I’m confident that there are many more.
The competing hypotheses, (hopefully) clarified:
Catalytic antibodies are structurally constrained and must function in the context of the immunoglobulin (Ig) fold.
If their prevalence is representative of the prevalence of function in sequence space, if we experimentally release them from that constraint by adding random sequence on each end, at the end of several selection cycles we should observe that enzymatic activities are significantly greater than those of the parent catalytic antibodies that were selected as antibodies.
If their prevalence is not representative of the prevalence of function in sequence space, if we experimentally release them from that constraint by adding random sequence on each end, at the end of several selection cycles we should observe that enzymatic activities are the same or lower than those of the parent catalytic antibodies that were selected as antibodies.
@T_aquaticus, granted pepsin is optimized for very acidic conditions. But most proteins aren’t. And I wasn’t talking about beta lactamase or the phage display papers.
A post was split to a new topic: The Design Meta-Scientific Hypothesis