Designer Proteins

[Nick R]

Recently, I've been very interested in the topic of completely human designed (de novo) proteins that are functional for a specific purpose, and decided to pose a question: If you could create any designer protein for a specific function, what would it's function be? 

[Nathan McCorkle]

I'd make high-value small-molecules... things like medicines, flavors, nanotech

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-Nathan

[Nick R]

Very smart. I was thinking about how great it would be if we had an enzyme that combined the gRNA guided dsDNA breaks of cas9 with retroviral integrase's powers of insertion. Guided, in vitro, single organism genetic engineering. I think that would be amazing. 

[Koeng]

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015364

I'm working with some of these right now. De novo proteins are extremely inefficient compared to the naturally evolved versions. Directed evolution is an interesting prospect.

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/2/groupllintronskarberg.pdf

Targeted reverse transcription exists. Gotta add Mg+ if you want integration in normal cells but it works fine in bacteria/mitochondria. Targetronics has 2 different ones you can use and sigma has quite a selection of premade vectors. 

If I am really interested in a new protein, usually I would try to see if something like it exists in nature. That will *always* be more efficient than a man-made protein (completely new ones that is, not just modified ones like GFP)

Anyway, on de novo I would really like a monocistronic ribosome (skip to overall to get past boring experimental sutff). I've thought about how this could be completed in E coli by fusing all the parts together and cutting it apart with TEV (in vivo TEV- http://www.ncbi.nlm.nih.gov/pubmed/15741334 ). Essentially, I would PCR out each E coli small ribosome proteins (~22). Overall, this is about 9450bp so it could even be synthesized. During this, I would remove all the BsaI sites with site directed mutagensis during the cloning (aka, SLiCE 2 parts together with a replaced codon). I would clone this into a vector used for BASIC cloning ( http://pubs.acs.org/doi/abs/10.1021/sb500356d ), because of the modularity. The modularity is important because later variants can be easily made. After this cloning reaction, I would have 23 parts (22 small ribo + TEV) which I would need to piece together. This would be accomplished in 2 steps, a ~4 part BASIC cloning producing 6 plasmids which then are goldengated together. The BASIC overlap sequence, a part which I can modularily insert a linker, will be either a RBS or a TEV site. After this, I would screen the genome by making deletions in ribosome genes by targeting Cas9 to either the BsaI sites I recoded (BsaI is GGTCTC, PAM is NGG) or on flanking sides, which then I would integrate KanR into. Hypothetically, my plasmid will contain the full gene under a normal RBS, so this deletion will occur. But if I can't get it to work with the TEV I know that making the protein monometric in this fashion will require more work. Overall I think it would be really cool to be able to express on a single RNA molecule the entire ribosome, T7 (to transcribe itself), and it's rRNA (tethered together). Good first step, I'd think, to a modular synthesizable genome.

There's Cathal's idea of DNA synthesis using enzymes, which honestly would be super cool. Essentially, you alternate light for each nucleotide and 'break' step where DNA nucleotides are added onto a chain on the basis of the order of the light you shine on the enzymes. 

After that, I'd probably just make medicines and such. Maybe an orthogonal ribosome to modularly create lots small peptides with lots of modifications. Expand the code so you could say, produce penicillin, ribosomally. 

-Koeng

[Cathal (Phone)]

Cell specific, gene specific promoter activation or repression. Imagine what you could do if you could pop a pill and upregulate just one gene by a scalar amount for a predictable timeframe?

My favourite application: activating retroviral genomes, so you could use in combination with HAART to clear residual HIV infection.

On 17 November 2015 22:28:02 GMT+00:00, Nick R <ratednf...@gmail.com> wrote:

Recently, I've been very interested in the topic of completely human designed (de novo) proteins that are functional for a specific purpose, and decided to pose a question: If you could create any designer protein for a specific function, what would it's function be? 

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Sent from my Android device with K-9 Mail. Please excuse my brevity.

[David Weichselbaum]

Like Koeng laid, artificial enzymes are very inefficient atm. If you make them from protein.

The problem is the insane amount of calculation time you need to pour into getting the secondary structure just right. And with an enzyme 1Å makes all the difference.

Now there are RNA and even DNA catalysts, but they lack the flexibility of proteins. There are reactions they just can't do.

A marriage of the very predictable structures formed by DNA origami and the versatility of peptide chemistry is the short term solution I think.

DNA-based binding motives for a substrate of choice is to be found using SELEX. Around that one would engineer the scaffold to hold functional peptides via DNA-binding domains. Naturally, a covalent binding would be better, maybe somebody has an idea for this.

The DNA part of the structure would be more durable than regular enzymes. Peptide parts could be replaced constantly.

This DNAzymes could be used in environments that are to harsh for normal proteins, like industrial synthesis under high pressures and temperatures. They could be used together with cleaning agents or in low-moisture environments. Extending the DNA scaffold one could crystallize it easily while preserving functionality. 

The reason you can't add metabolic functionality to a body is the reaction of its immune system to foreign proteins. DNAzymes could be deployed in the blood stream to serve custom reactions, given most of the peptide domains are derived from humans. 

[Cathal (Phone)]

If you're not already familiar with Peptide Nucleic Acids, Mister Cherrytree, I recommend reading up. :)

Because they're based on an amino backbone, they have higher binding affinity to DNA than DNA does, and you can conjugate peptides or proteins to them easily, albeit only in vitro.

[John Griessen]

On 11/17/2015 04:28 PM, Nick R wrote:
> If you could create any designer protein for a specific function, what would it's function be?

produce enzymes that:

bond lignin at low temperatures.

bond cellulosics to silicates at low temperatures, near neutral pH.

[Nick R]

Oh man those all sound like awesome concepts. Hopefully the technology will advance enough relatively quickly. 

On Tuesday, November 17, 2015 at 2:28:02 PM UTC-8, Nick R wrote:

[Yuriy]

Who knows, maybe someone will pull this one off.

Non-oxidative stress driven, gene (codon) amelioration mechanism. Optimization according to the chassis.