# Beta-Lactamase, Antibody Enzymes, and Sequence Space

**URL:** <https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271>\
**Category:** Conversation\
**Tags:** Science, Design\
**Created:** [January 31, 2019, 9:15pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271 "2019-01-31T21:15:32Z")\
**Posts on this page:** 20\
**Page:** 6

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**Author:** ![T\_aquaticus](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/t_aquaticus/32/1249_2.png) [@T\_aquaticus](https://discourse.peacefulscience.org/u/T_aquaticus)\
**Post date:** [February 4, 2019, 3:56pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/101 "2019-02-04T15:56:04Z")

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> [@The Extra Face in Mount Rushmore](https://discourse.peacefulscience.org/t/the-extra-face-in-mount-rushmore/4067/205):
>
> I challenge any of you to obtain B-lactam activity from a true soluble enzyme, not an antibody fragment pinned to phage.

@Agauger  
Already done.

> **[Catalytic mechanism of an abzyme displaying a beta-lactamase-like activity -...](https://link.springer.com/article/10.1385/ABAB:83:1-3:163)**
>
> A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site. We have previously...

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

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<div class="post-metadata">

**Author:** ![T\_aquaticus](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/t_aquaticus/32/1249_2.png) [@T\_aquaticus](https://discourse.peacefulscience.org/u/T_aquaticus)\
**Post date:** [February 4, 2019, 4:04pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/102 "2019-02-04T16:04:22Z")

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> [@The Extra Face in Mount Rushmore](https://discourse.peacefulscience.org/t/the-extra-face-in-mount-rushmore/4067/261):
>
> So what does the 1 in 10^10 represent 1 in 10^10 phage screened? This must mean that the mutagenesis is very focus on a small area, so as to sample as wide a number of variants as possible in the region of substrate binding. If so, and I will go back and check the methods, that is another reason the number is so low. Fewer residues to be sampled.

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

> **[V(D)J recombination](https://en.wikipedia.org/wiki/V(D)J_recombination)**
>
> V(D)J recombination is the mechanism of somatic recombination that occurs only in developing lymphocytes during the early stages of T and B cell maturation. It results in the highly diverse repertoire of antibodies/immunoglobulins and T cell receptors (TCRs) found in B cells and T cells, respectively. The process is a defining feature of the adaptive immune system.
> V(D)J recombination in mammals occurs in the primary lymphoid organs (bone marrow for B cells and thymus for T cells) and in a nearl...

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.

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**Author:** ![Agauger](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/agauger/32/144_2.png) [@Agauger](https://discourse.peacefulscience.org/u/Agauger)\
**Post date:** [February 5, 2019, 5:48am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/103 "2019-02-05T05:48:04Z")

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> [@The Extra Face in Mount Rushmore](https://discourse.peacefulscience.org/t/the-extra-face-in-mount-rushmore/4067/274):
>
> 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.

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?

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**Author:** ![T\_aquaticus](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/t_aquaticus/32/1249_2.png) [@T\_aquaticus](https://discourse.peacefulscience.org/u/T_aquaticus)\
**Post date:** [February 7, 2019, 10:04pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/104 "2019-02-07T22:04:57Z")

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> [@Agauger](#):
>
> 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?

@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.

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**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 7, 2019, 10:07pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/105 "2019-02-07T22:07:36Z")

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> [@T\_aquaticus](#):
>
> 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.

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.

> [@Agauger](#):
>
> Are you saying that is how abenzymes are made, by immunization and clonal selection? Or is it all done in vitro somehow?

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

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**Author:** ![Agauger](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/agauger/32/144_2.png) [@Agauger](https://discourse.peacefulscience.org/u/Agauger)\
**Post date:** [February 8, 2019, 4:48am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/106 "2019-02-08T04:48:16Z")

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

> [@swamidass](#):
>
> > Are you saying that is how abenzymes are made, by immunization and clonal selection? Or is it all done in vitro somehow?
> 
> It depends on the precise method used, of course. There are many ways to do it

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**Author:** ![Agauger](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/agauger/32/144_2.png) [@Agauger](https://discourse.peacefulscience.org/u/Agauger)\
**Post date:** [February 8, 2019, 5:10am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/107 "2019-02-08T05:10:05Z")

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@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.

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**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 8, 2019, 5:18am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/108 "2019-02-08T05:18:02Z")

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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.

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**Author:** ![Agauger](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/agauger/32/144_2.png) [@Agauger](https://discourse.peacefulscience.org/u/Agauger)\
**Post date:** [February 8, 2019, 5:47am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/109 "2019-02-08T05:47:51Z")

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@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.

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**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 8, 2019, 5:49am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/110 "2019-02-08T05:49:09Z")

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> [@Agauger](#):
>
> You misunderstand. If they immunized, it was in vivo, and there was plenty of selection _before_ harvest for making the antibody producing clones.

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.

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**Author:** ![T\_aquaticus](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/t_aquaticus/32/1249_2.png) [@T\_aquaticus](https://discourse.peacefulscience.org/u/T_aquaticus)\
**Post date:** [February 8, 2019, 4:08pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/111 "2019-02-08T16:08:53Z")

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> [@Agauger](#):
>
> 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.

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.

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**Author:** ![Agauger](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/agauger/32/144_2.png) [@Agauger](https://discourse.peacefulscience.org/u/Agauger)\
**Post date:** [February 8, 2019, 8:54pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/112 "2019-02-08T20:54:07Z")

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@T_aquaticus

> [@T\_aquaticus](#):
>
> 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.

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

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**Author:** ![Art](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/art/32/3805_2.png) [@Art](https://discourse.peacefulscience.org/u/Art)\
**Post date:** [February 8, 2019, 9:36pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/113 "2019-02-08T21:36:11Z")

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> [@Agauger](#):
>
> It has to do with the density of mutation and the preselection. But I am still working through this.

@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.

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**Author:** ![Mercer](https://avatars.discourse-cdn.com/v4/letter/m/e274bd/32.png) [@Mercer](https://discourse.peacefulscience.org/u/Mercer)\
**Post date:** [February 8, 2019, 10:42pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/114 "2019-02-08T22:42:51Z")

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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.

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**Author:** ![Mercer](https://avatars.discourse-cdn.com/v4/letter/m/e274bd/32.png) [@Mercer](https://discourse.peacefulscience.org/u/Mercer)\
**Post date:** [February 8, 2019, 10:58pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/115 "2019-02-08T22:58:15Z")

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> [@Agauger](#):
>
> If they immunized, it was in vivo, and there was plenty of selection _before_ harvest for making the antibody producing clones.

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

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**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 8, 2019, 11:18pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/116 "2019-02-08T23:18:53Z")

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> [@Mercer](#):
>
> In this paper, one library, which yielded **two enzymatically active antibodies** , was from an unimmunized source, so your point is moot.

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.

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**Author:** ![Rumraket](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/rumraket/32/9328_2.png) [@Rumraket](https://discourse.peacefulscience.org/u/Rumraket)\
**Post date:** [February 8, 2019, 11:25pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/117 "2019-02-08T23:25:14Z")

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> [@swamidass](#):
>
> 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.

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.

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<div class="post-metadata">

**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 8, 2019, 11:28pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/118 "2019-02-08T23:28:11Z")

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> [@Rumraket](#):
>
> Behe actually has come up with an answer to this.

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).

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<div class="post-metadata">

**Author:** ![Mercer](https://avatars.discourse-cdn.com/v4/letter/m/e274bd/32.png) [@Mercer](https://discourse.peacefulscience.org/u/Mercer)\
**Post date:** [February 9, 2019, 12:11am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/119 "2019-02-09T00:11:05Z")

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> [@Agauger](#):
>
> > Their clinical utility is completely irrelevant to the point we’re discussing.
> 
> This remark baffles me. Clinical utility? Their utility _as enzymes_ has everything to do with what we are discussing. Do they or are they capable of making good Beta lactamase enzymes. The answer appears to be no.

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

> [@How Does Tokuriki 2009 Affect Conclusions from Axe 2004?](https://discourse.peacefulscience.org/t/how-does-tokuriki-2009-affect-conclusions-from-axe-2004/2774/3):
>
> Doug [Axe] did not want to measure where buffering was taking place, neither did he want to measure during exponential decline. He wanted to measure at the threshold of enzyme activity, because he was trying to quantify the number of changes to go from no function to function. This is the transition boundary that is of interest to evolutionary biologists.

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?

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<div class="post-metadata">

**Author:** ![swamidass](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/swamidass/32/3_2.png) [@swamidass](https://discourse.peacefulscience.org/u/swamidass)\
**Post date:** [February 12, 2019, 6:06pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/120 "2019-02-12T18:06:51Z")

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6 posts were split to a new topic: [Draper’s Paper on Irreducible Complexity](https://discourse.peacefulscience.org/t/drapers-paper-on-irreducible-complexity/4477)

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