Antibody Enzymes and Sequence Space

Design
Science

#101

@Agauger
Already done.

It is a whole IgG molecule that is soluble and has beta-lactamase activity.


#102

@Agauger
It is important to remember how antibody genes come to be.

There are many different short DNA sequences that are shuffled and then stitched together during B-cell maturation. There are also indels that change can change the reading frame within those fragments. What you get is billions of antibodies with different Fab segments. This happens while you are still in the womb. You are born with a massive library of antibodies, and when one of those B-cell bound antibodies binds to an appropriate antigen that B-cell starts dividing and pumping out more of that antibody. You get positive selection of pre-existing antibodies.


(Ann Gauger) #103

I know that how it usually happens. Are you saying that is how abenzymes are made, by immunization and clonal selection? Or is it all done in vitro somehow?


#104

@Agauger
I can’t get past the paywall, but I would assume that they either harvested serum or B-cells after immunizing in vivo. It would be way more difficult to do the work in vitro, and there are multiple streamlined methods for either polyclonal or monoclonal antibody production in vivo.

Immunization will stimulate the B-cell lineage with the antibody for that antigen. This will result in clonal expansion and an increase of that antibody in serum. Both of these features makes it easier to find the antibody you are looking for in serum and in harvested B-cell populations.


(S. Joshua Swamidass) #105

This is what the paper says:

Biozzi mice were immunized with 200 µg of 7AF9 according to standard procedures. Monoclonal antibodies (MAbs) were produced and screened for both 7AF9 recognition and benzylpenicillin hydrolysis ability.

As I’ve explained before, simple enzymes are just a special case of “binding.” From simple enzymes, it is not hard to see how they can become more complex.

It depends on the precise method used, of course. There are many ways to do it.


(Ann Gauger) #106

I need to see the paper itself. Still catching up from the trip.


(Ann Gauger) #107

@swamidass @Mercer
So I am just revisiting this abenzyme story. The paper with the phage display. I have to go back and look it up, but did they make the antibodies by immunization with the drug target, then recover cells and make monoclonal antibodies?All this before putting the antibodies on phage? I have to look at the methods. Because it seems to me there are many rounds of selection happening in vivo that aren’t being counted if that is how it is done. Now I have to find the paper again.


(S. Joshua Swamidass) #108

I forgot the details, but I do recall @T_aquaticus and @mercer saying there was just one round of selection on this one. Phage display is not an in vivo system.


(Ann Gauger) #109

@swamidass

You misunderstand. If they immunized, it was in vivo, and there was plenty of selection before harvest for making the antibody producing clones. It’s been ages since I have done it, but making antibodies involves injecting a mouse or rabbit (or whatever) with the antigen, often multiple times. The body does the work of selecting and amplifying the best clones.


(S. Joshua Swamidass) #110

There are two papers. One was a phage study, one was an in vivo mouse study. I did not misunderstand. You didn’t specify which paper you were asking about. Which one? There are two under discussion.


#111

Those clones were created before being exposed to the antigen. In vivo, selection is occurring among already existing antibodies. More to your point, there is somatic hypermutation during clonal expansion, but I don’t see how the mechanism of random mutations detracts from our argument. In fact, it adds to it. If a randomly assembled Fab followed by random mutation and selection can produce a protein with beta-lactamase activity it would seem to be a serious challenge to Axe’s previous statements about the rarity of beta-lactamase activity in protein sequence space.


(Ann Gauger) #112

@T_aquaticus

It has to do with the density of mutation and the preselection. But I am still working through this.


(Arthur Hunt) #113

@Agauger, I seriously doubt that you will be able to “find”, in the concepts of mutation density and preselection, the probabilistic resources to reconcile the abzyme field with Axe’s claims.


(John Mercer) #114

It was a mixture of libraries. Two catalytic antibodies came from the unimmunized library, with no rounds of selection. ZERO preselection. That’s a very important point.


(John Mercer) #115

In this paper, one library, which yielded two enzymatically active antibodies, was from an unimmunized source, so your point is moot.


(S. Joshua Swamidass) #116

Actually, it was two antibodies with irreducibly-complex activity. Remove the phage proteins, and the activity vanishes. Remove the antibody, and the activity vanishes too. Interlocking parts, both of which are necessary for activity.


(Mikkel R.) #117

Behe actually has come up with an answer to this. It’s a silly one, but an answer nevertheless. Behe essentially argues in Darwin’s Black Box that simple irreducibly complex functions can evolve, by which he means systems with 2-3 components, but that as the number of components of the system increases, he argues, the probability of evolving it goes down.

It is obvious what the issue with this kind of argument is. Essentially Behe has sort of argued that his concept only really takes effect once the complexity of the system is beyond what we should normally expect to see happening in human lifetimes.

Sure, we can see a 2-3 component system evolve in some experiment, but what about a 20, or 50 component system.

With this very arbitrary limitation, Behe has made sure to put the goalposts beyond the results of what could be reasonably expected to be observed in an experiment happening in a couple of decades of study. In this way Behe will always be able to argue that more complex systems have never been observed evolving, and that only “modest” and “simple” results are achieved in lab experiments.

It is difficult to resist suspicion that he did this intentionally just so he could fall back on the age-old creationist “observational” vs “historical” science objection.


(S. Joshua Swamidass) #118

Sure, but it still fits his definition of Irreducibly Complex (IC1), even if it only requires two parts. This is an irreducibly complex enzyme fished out of a random pool of phages, without any selection.

At the same time, I should point out, we don’t know precisely how many pieces of the phage are required for the enzyme to work. So it could be much more than 2. I’d guesstimate it would require more than 3 parts (but I could be totally wrong here).


(John Mercer) #119

I am puzzled, because that stands in stark contrast to what you wrote a while ago:

So are you now saying that catalytic antibodies do not fit this interest in the threshold of enzyme activity, or that you do not share Doug’s interest in the boundary?


(S. Joshua Swamidass) split this topic #120

6 posts were split to a new topic: Draper’s Paper on Irreducible Complexity