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Chemical Biology

Phage-Derived Catalysts

I’ve been enjoying this recent paper in JACS, but then I always like to see intersections of molecular biology with organic synthesis. The authors, from CUNY and the University of Strathclyde, are using a phage display library to see if they can come up with displayed peptide combinations that are catalytic. Specifically, they’re trying to form an amide bond from a primary amine and a methyl ester, which at RT in water is going to be a pretty slow process. (But as numerous enzymes show, it’s certainly capable of being accelerated).
The phages display five copies of a 12-mer peptide sequence, and they have about 3 billion different sequences in the library. That alone is why I’m always interested in phage display, DNA-encoded libraries, and the like: the sheer number of combinations that can be worked with. There aren’t three billion reported organic compounds in the history of the human race – maybe one-twentieth that number. But there are three billion in that Eppendorf vial over there. The problem is to tell the few that you want from the billions that you don’t, and that’s where the molecular biology really kicks in.
In this case, though, the authors used an ingenious trick to find the phages that would catalyze their reaction. The reacting partners were Fmoc-threonine and leucine methyl ester, and the product peptide is so insoluble that it tends to aggregate around the business end of the phage that’s producing it. Careful centrifugation of the mixture, then, will give you a heavier band of phage(s) with more apparent weight, and treatment of those with subtilisin cleaves all those methyl esters and allows the stuck peptides to be washed off. Those phages are then taken back into E. coli and amplified for another round, and after a couple of cycles, they sequenced samples of the phages that had been selected.
To control for possible things happening to that methyl ester along the way, they did the same sorts of experiment with leucineamide, this time using acetonitrile/water to wash off the formed peptides from the phages, rather than hydrolyzing the methyl esters. Comparing the sequences from all these selections shows. . .well, a lot of variation. There aren’t any obvious similarities, either among the phages identified in the two experiments, or between the ester/amide ones. But the selected phages do tend to catalyze amide formation; that much seems clear. One difficulty is that the amide formation appears self-limiting, since the aggregating product gums up the region doing the catalysis (the differences in aggregation in that area between active phages and inactive ones can be seen in electron microscopy images).
So you can apparently find catalytically active proteins, even just in repeated dodecapeptide displays. And there are apparently a number of different ways for them to be active (thus the sequence variability). This work reminds me of a phage-display paper I blogged about last year, where the authors were searching for molecular recognition sequences. (Note, though, that as pointed out in the comments, the binding data in that paper are not as compelling as they should be).
So we have some evidence that phage-display peptides could have applications in organic chemistry, but there’s nothing conventionally useful yet. The techniques being used are still probably not exerting enough selection pressure. In the current paper, the selected phages definitely stand out from random background, but they’re not exactly artificial ribosomes, either. Maybe it’s the fairly simple nature of the phage display, or the product inhibition. I can’t help but think that there are useful and interesting things out there in those crazy billions of phage-derived peptides, although it’s worth remembering that the three billion peptides in this experiment are still only about one-millionth of the possible number of 12-mer sequences. We still need better ways to produce, select, and evaluate them.
Update: Paul D. in the comments put me onto a company I hadn’t been aware of, Siluria. They’ve been using phage display as a screening platform to product new inorganic catalysts, a sort of combinatorial biomineralization process, that seems to be yielding results. Here’s an article on them, and here’s a note on the progress in their methane-to-ethylene process.

8 comments on “Phage-Derived Catalysts”

  1. Paul D. says:

    The phage display technique has been used by a company named Siluria to make industrial catalysts; in particular, a catalyst for the oxidative coupling of methane (OCM) to ethylene:
    2 CH4 + O2 -> C2H4 + 2 H2O
    Methane is a low cost feedstock, so this is potentially quite lucrative.
    They mix phages with various combinations of inorganic metal compounds and heat to drive off the organic stuff. The phage surfaces pattern the inorganic ions, altering the local chemical environment of the catalyst in ways that would be difficult to predict, but that can be found by selection and experimentation. The phages also cause the catalyst to have an attractive “nanowire” consistency with high surface area.
    The result is a catalyst with high activity and selectivity, at fairly mild (compared to previous efforts) conditions, and with long lifespan. They’ve recently finished raising series D funding.
    Siluria went through 100,000 or more different formulations using high throughput screening. I could see this approach having an impact in many areas of industrial chemistry. Siluria also has a catalyst for conversion of the ethylene to liquid fuels, for a one-reactor “methane to gasoline” process.

  2. luysii says:

    Phage display can also be used to find out what sequences a protease can cut. PNAS 111 E4148 – 4155 ’13 exposed all possible 6 amino acid peptides (64,000,000 == 20^6) on phage to see what a variety of matrix metalloproteases (MMPs) could actually cut. That was pretty interesting, but even more interesting was their work on what determined catalytic specificity. They found that 57 discontinuous amino acids on the front face of the MMPs determined catalytic preference. Changing them shifted what the MMP would cut.
    While 64,000,000 is a large number and 3 billion is an even larger number, 3 billion is a small fragment of the 20^12 possible 12 amino acids (actually 1/4,096,000) of the possibilities. The proteins making us up are a tiny, tiny fraction of the possibilities in protein space.
    A while back I posted a back of the envelope calculation of how many different proteins you could make assuming the entire mass of the earth was made entirely of C, H, O, N, S. All you needed to do was just make one molecule of each. The results were pretty surprising.
    See http://luysii.wordpress.com/2009/12/20/how-many-proteins-can-be-made-using-the-entire-earth-mass-to-do-so/

  3. SP says:

    I forget if you already highlighted this, but it’s the way to go for phage evolution- http://www.ncbi.nlm.nih.gov/pubmed/21478873

  4. Derek Lowe says:

    #3, SP – I really enjoyed that paper, and kept meaning to blog on it, but never did. I see the Liu et al. have improved the method this year, though, so I’ll blog on that!

  5. Anonymous says:

    And to think some of this work was done in Glasgow – the setting for the film Trainspotting!

  6. Anonymous says:

    bl**dy f****ng h*ll
    this movie was set in edinburgh, not with the soap haters…

  7. Anonymous says:

    Same thing

  8. Daen says:

    My former employer, Nuevolution in Copenhagen, which uses a DNA-encoded library technology they call Chemetics, got started through one of its co-founders working with phage display at Scripps and Genentech. Richard Lerner’s work on phage display and DNA-encoded combichem was certainly a big inspiration in the early days.

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