# 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:** 7

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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 12, 2019, 6:07pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/121 "2019-02-12T18:07:34Z")

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A post was merged into an existing topic: [Draper’s Paper on Irreducible Complexity](https://discourse.peacefulscience.org/t/drapers-paper-on-irreducible-complexity/4477/7)

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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 14, 2019, 10:28am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/122 "2019-02-14T10:28:31Z")

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

How’s it going?

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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 28, 2019, 5:19pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/123 "2019-02-28T17:19:09Z")

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@Mercer

As I understand it( and I still don’t have the paper(s)describing how they were made and characterized)the catalytic antibody you are describing came from an animal that was not immunized. Was it ever treated with a penicillin

Or related antibiotic?

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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 28, 2019, 5:40pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/124 "2019-02-28T17:40:40Z")

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@Agauger, I just sent you and Brian Miller those three papers.

And @Mercer, @agauger is correct, the paper you were pointing to pulled the antibiodies from some immunized and some non-immunized animals. Can you determine which of the antibodies came from which source? Or can you help me understand my misreading of the paper? Or can you produce a different paper that demonstrates your point?

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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 28, 2019, 6:20pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/125 "2019-02-28T18:20:38Z")

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I wonder what the argument is going to be here. Are we to believe that a previously immunized animal in fact generated ~10^77 different proteins, and that is why a beta-lactamase active abzyme was found?

Even if the animal generated something like 10^20 different proteins and only found one, that’s still _fifty seven orders of magnitude_ more frequent than most ID proponents who’ve heard of Axe’s work believes functional proteins are found in sequence space.

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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 28, 2019, 6:36pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/126 "2019-02-28T18:36:26Z")

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Let’s just get the facts straight before gaming out what will happen from here.

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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:** [March 1, 2019, 12:34am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/127 "2019-03-01T00:34:55Z")

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> [@Agauger](#):
>
> As I understand it( and I still don’t have the paper(s)describing how they were made and characterized)

It has been provided. Most recently, it was provided in post #16 above in a reply to you:  
[https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14012](https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14012)

It appears to me that you don’t see what you don’t want to see. You’ve also not acknowledged that there are more than 5000 other papers in the field.

> [@Agauger](#):
>
> the catalytic antibody you are describing came from an animal that was not immunized.

Two of them did.

_In the present study, we report the construction of a phage display scFv library of size 2.7 × 109, from the classical murine strains Balb/C (healthy) and the SJL/J strain (susceptible to developing autoimmune disease), which has previously shown to express higher levels of catalytic antibodies [29](https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14012#febs14012-bib-0029), [30](https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14012#febs14012-bib-0030). This library represents four different IgG immune repertoires: (a) healthy and **nonimmunized** , (b) healthy and immunized with KLH‐conjugated penam sulfone hapten, (c) autoimmune prone and **nonimmunized** , and (d) autoimmune prone and immunized. **The repertoires are identifiable via a novel ‘restriction bar‐coding’** technique, providing the first reported example of such methodology, in order to perform 2D screening._

> [@Agauger](#):
>
> Was it ever treated with a penicillin
> 
> Or related antibiotic?

What would that have to do with anything?

And why are you pretending that this is about a single paper, not 5000?

It doesn’t have to be done by phage display.

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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:** [March 1, 2019, 12:36am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/128 "2019-03-01T00:36:01Z")

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@mercer, it seems the some of the animals in the study are immunized. Do you see that? Did I miss something?

Do we know of the active antibodies came from the non immunized or immunized rats?

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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:** [March 1, 2019, 9:05am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/129 "2019-03-01T09:05:26Z")

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There were active ones from both, as shown above.

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**Author:** ![cwhenderson](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/cwhenderson/32/72_2.png) [@cwhenderson](https://discourse.peacefulscience.org/u/cwhenderson)\
**Post date:** [March 1, 2019, 3:08pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/130 "2019-03-01T15:08:08Z")

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> [@swamidass](#):
>
> Do we know of the active antibodies came from the non immunized or immunized rats?

I’m not certain this matters. The VDJ joining (and other mechanisms) would have all occurred in the B cell precursors prior to any immunization, right?

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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:** [March 1, 2019, 4:22pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/131 "2019-03-01T16:22:25Z")

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> [@cwhenderson](#):
>
> I’m not certain this matters. The VDJ joining (and other mechanisms) would have all occurred in the B cell precursors prior to any immunization, right?

If a B-cell binds to an antigen and is given the signal to proliferate there is a process of somatic hypermutation that occurs in the variable region of the antibody. Mutations can increase binding which also increases the proliferation signal. Antibodies evolve over time with selection favoring an increase in binding. This process doesn’t produce completely new antibodies, but it does tweak the sequences that were originally produced by VDJ recombination. Therefore, I think it is fair to ask if these antibodies came from immunized animals since immunization can change the immunoglobulin library that they are looking at.

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**Author:** ![cwhenderson](https://sea2.discourse-cdn.com/flex016/user_avatar/discourse.peacefulscience.org/cwhenderson/32/72_2.png) [@cwhenderson](https://discourse.peacefulscience.org/u/cwhenderson)\
**Post date:** [March 1, 2019, 5:53pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/132 "2019-03-01T17:53:51Z")

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Makes sense, thanks for the clarification.

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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:** [March 1, 2019, 6:04pm UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/133 "2019-03-01T18:04:52Z")

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One point that subverts the idea of this being caused by prior immunization is that the enzyme is IC1, requiring the phage particle, not functional on its own.

@Rumraket notes earlier that even if it was immunized, that doesn’t explain how the immune system searched a 10^77 space, as Axe/@Agauger would predict. Perhaps you could argues 10^15 or so, but we are very far from the ID prediction.

As @mercer notes, this is not the only paper we can go to. There are many many more. So we can work through the next paper after this is digested.

This seems to be a major problem for ID theory, which has relied on the correctness of Axe’s hypothesis. It seems this data invalidates the hypothesis. I am very interested to see @Agauger and @bjmiller’s response.

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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:** [March 2, 2019, 2:53am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/134 "2019-03-02T02:53:32Z")

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My question is, how large is the epitope universe? How many unique epitomes are there. More than 10^10? Many more? Does it not surprise you that out of 5 clones characterized , 3 were from the non-immunized library? Would you therefore expect other proteins also have abzymes innate in naive animals?

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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:** [March 2, 2019, 3:01am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/135 "2019-03-02T03:01:55Z")

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I think there is something different, not comparable between axe’s Method, Dill’S method etc and the abzyme work. For one the the immune repertoire starts out stable and folded. Axe’s calculations as I recall, started without that because he was considering the universe of protein sequence as his starting point. I am writing this on an airplane waiting to leave so pardon the haste.

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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:** [March 2, 2019, 3:56am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/136 "2019-03-02T03:56:19Z")

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> [@Agauger](#):
>
> My question is, how large is the epitope universe? How many unique epitomes are there. More than 10^10?

Less. 10^8.

> [@Agauger](#):
>
> Does it not surprise you that out of 5 clones characterized , 3 were from the non-immunized library?

No.

> [@Agauger](#):
>
> Would you therefore expect other proteins also have abzymes innate in naive animals?

Yes, but “proteins also have abzymes” is extremely unclear.

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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:** [March 2, 2019, 3:58am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/137 "2019-03-02T03:58:13Z")

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> [@Agauger](#):
>
> I think there is something different, not comparable between axe’s Method, Dill’S method etc and the abzyme work.

Yes. Axe’s was horribly sloppy and dealt with an N of 1. He didn’t measure enzyme activity. He started with a ts mutant.

Most telling, he didn’t follow up his outlier result.

> [@Agauger](#):
>
> Axe’s calculations as I recall, started without that because he was considering the universe of protein sequence as his starting point.

How could they, when he was starting with a ts mutant of a single protein?

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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:** [March 2, 2019, 4:07am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/138 "2019-03-02T04:07:48Z")

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> [@Agauger](#):
>
> I think there is something different, not comparable between axe’s Method, Dill’S method etc and the abzyme work. For one the the immune repertoire **starts out stable and folded**.

This is a distinction without significance for three reasons:

1. A stable fold is not a requirement for an enzyme in the first place. Intrinsically disordered proteins are likely an important initial state for de novo enzymes, that are subsequently tightened up into a stable fold by positive selection. For an IDP to work as an enzyme, it needs _one_ groove in the protein to be in the right configuration _some_ of the time. That isn’t too hard to do, and might even be easier for an IDP than a stable protein.

2. It is fairly easy to get a stable fold. Right now, protein prediction algorithms are good enough to study this. Put some random sequences in. See how often stable folds arise. It is quite easy. (I think @T_aquaticus has done this before).

3. An antibody is essentially a stable fold with a variable groove or pocket. Imagine a protein has one function, and is maintained by negative selection to be a stable fold. An “unused” groove of the protein can be varied by neutral evolution till it finds the function. Antibody evolution mimics this process. Add in the Real Time-Evolution mechanism you just wrote about (duplication-then-divergence), and you have a new enzyme, with a totally new function.

So for those three reasons, I do not think there is reason to doubt the relevance of these studies.

[EDIT: Removed discussion on CCC. It’s too late to get this straight.]

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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:** [March 2, 2019, 4:27am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/139 "2019-03-02T04:27:01Z")

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OK,

[My question to Mercer]  
My question is, how large is the epitope universe? How many unique epitomes are there. More than 10^10?  
[/quote]

> [@Mercer](#):
>
> Less. 10^8.

Let me clarify. By epitope you mean the things the antibodies can recognize, and it’s by definition going to be a different set for every organism, right. I have 10^8, You hav a different 10^8 etc.There are 10^8 antibodies and theach have an epitope  
So how many different protein shapes are there in the protein universe? More than 10^8. How many different proteins are there? A single amino acid change can change antibody recognition. Just think flu virus.  
This observation gives me pause. That one protein should be represented so frequently in so large a universe argues to me that something has been overlooked

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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:** [March 2, 2019, 4:29am UTC](https://discourse.peacefulscience.org/t/beta-lactamase-antibody-enzymes-and-sequence-space/4271/140 "2019-03-02T04:29:03Z")

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@swamidass what is a CCC?

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