Enzymatic protein synthesis without DNA and without RNA

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Bryan Bishop

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Dec 25, 2014, 3:25:09 PM12/25/14
to enzymatic...@googlegroups.com, Bryan Bishop, Cathal Garvey
Here's an idea. Since no particular progress has been made on the problem of enzymatic DNA synthesis, what about focusing on enzymatic synthesis of proteins instead?

Currently, in vitro protein synthesis protocols require DNA or mRNA.... I don't think this is impossible to override, though. This would require either hacking a ribosome or hacking tRNA synthetase. Ultimately the protocol would probably end up with steps like "select one of 20 vials of tRNAs already attached to tRNA and amino acids, insert into ribosome reaction".

Antibiotics already cause mistranslation in ribosomes, so those mechanisms could be studied to determine how easy or difficult it may be to cause only mistranslation in a ribosome.

Additionally, some method of capping or blocking a ribosome would be necessary, so that after delivering an amino acid to the ribosome that no other tRNA synthetase could make a delivery. Next, the protocol would have to call for a wash step to remove the excess tRNA synthetase and amino acids, next decap, then proceed on to the next step. These wash/wait steps would have to be compatible with the ribosome.

The advantage of this method is that unlike oligonucleotide synthesis, large proteins can be constructed. Personally I would find this most useful for the construction of DNA polymerases, which so far cannot be directly synthesized in one molecule constructed by the phosphoramidite method. by experimenting with the amino acid design of polymerases, perhaps more efficient synthesis procedures can be made. But in the mean time, an effective cell-free, in vitro protein synthesis would be available without DNA and without RNA (or rather, without special/synthesized DNA).

What do you think?

Bryan Bishop

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Dec 25, 2014, 3:51:21 PM12/25/14
to enzymatic...@googlegroups.com, Bryan Bishop, Cathal Garvey
On Thu, Dec 25, 2014 at 2:25 PM, Bryan Bishop <kan...@gmail.com> wrote:
Here's an idea. Since no particular progress has been made on the problem of enzymatic DNA synthesis, what about focusing on enzymatic synthesis of proteins instead?

Similar to using TdT in oligonucleotide synthesis, there may be some way to use an enzyme in solid-phase peptide synthesis (instead of a completely-enzymatic protein synthesis): http://en.wikipedia.org/wiki/Peptide_synthesis#Solid-phase_synthesis

Cathal (Phone)

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Dec 25, 2014, 5:26:25 PM12/25/14
to Bryan Bishop, enzymatic...@googlegroups.com
How about this: degenerate mRNA nucleotides and orthologous tRNAs, so an arbitrary "next triplet" can be loaded with a chosen amino? To avoid repeats have two incompatible triplets staggered and two sets of 21 aminos per triplet. Feed charged tRNA stepwise, wash between. No modified ribosome required, minimal modified tRNAs.
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Bryan Bishop

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Dec 25, 2014, 5:43:13 PM12/25/14
to Cathal Garvey, Bryan Bishop, enzymatic...@googlegroups.com
On Thu, Dec 25, 2014 at 4:19 PM, Cathal Garvey <cathal...@cathalgarvey.me> wrote:
How about this: degenerate mRNA nucleotides and orthologous tRNAs, so an arbitrary "next triplet" can be loaded with a chosen amino? To avoid repeats have two incompatible triplets staggered and two sets of 21 aminos per triplet. Feed charged tRNA stepwise, wash between. No modified ribosome required, minimal modified tRNAs.

Could you expand on this? Especially the staggering and two sets of 21 aminos per triplet.

Dan Bolser

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Dec 25, 2014, 5:57:37 PM12/25/14
to enzymatic...@googlegroups.com, Bryan Bishop, Cathal Garvey

If I guess correctly, the idea is to have a generic AAAGGGAAAGGG... template mRNA, then a controlled way to feed complementarity tRNAs loaded with arbitrary aminos.

The question is, how easy / cheap is it to construct such loaded tRNAs?

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Bryan Bishop

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Dec 25, 2014, 6:06:04 PM12/25/14
to Dan Bolser, Bryan Bishop, enzymatic...@googlegroups.com, Cathal Garvey
On Thu, Dec 25, 2014 at 4:57 PM, Dan Bolser <dan.b...@gmail.com> wrote:

If I guess correctly, the idea is to have a generic AAAGGGAAAGGG... template mRNA, then a controlled way to feed complementarity tRNAs loaded with arbitrary aminos.


Specifically: since you know the mRNA sequence, you would know which tRNA synthetase to use next, based on a batch of tRNA synthetases with complementary tRNAs (complementary to the known mRNA sequence)?

Dan Bolser

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Dec 25, 2014, 6:11:23 PM12/25/14
to Bryan Bishop, Cathal Garvey, enzymatic...@googlegroups.com

Yeah. With zero noise, you could even just use AAAAAAAAAAA, but I guess alternation reduces error?

Bryan Bishop

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Dec 25, 2014, 9:41:54 PM12/25/14
to enzymatic...@googlegroups.com, Bryan Bishop, Cathal Garvey
On Thu, Dec 25, 2014 at 2:51 PM, Bryan Bishop <kan...@gmail.com> wrote:
Similar to using TdT in oligonucleotide synthesis, there may be some way to use an enzyme in solid-phase peptide synthesis (instead of a completely-enzymatic protein synthesis): http://en.wikipedia.org/wiki/Peptide_synthesis#Solid-phase_synthesis

Right.... looks like that exists:

Peptide synthesis: chemical or enzymatic (2006)

"""
Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective.
"""

and:

"""
Chemical synthesis is a viable technology for the production of small and medium size peptides ranging from about 5 to 80 residues (Kimmerlin and Seebach, 2005). Enzymatic synthesis is more restricted and has been hardly applied for the synthesis of peptides exceeding 10 residues.
"""

Looks like nobody has investigated using enzymes to synthesize larger peptides?

Here's an older paper that seems to be early in investigating the possibility of enzymatic deprotection in peptide synthesis:

Enzymes as reagents in peptide synthesis: enzymatic removal of amine protecting groups (1975)
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