3 ms·

The relevant limulus proteins can be expressed in vitro. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302

The relevant limulus proteins can be expressed in vitro. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/

jfarlow 9y ago
It looks like (very much like insulin production in vitro) the proteins involved here are highly modified after translation. Some proteins are usable right after they are produced by the ribosome, some require extensive modification to be useful. It looks like Factor-C [1] needs substantial posttranslational by machinery that is not present to the organisms commonly used for industrial protein production. So not only do you have to produce the protein synthetically, but you also have to produce the modification machinery synthetically. An additional challenge is that the readout is at the bottom of a signal-amplified cascade [2]. And that cascade magnifies any error you have at the top (which is where that finicky Factor-C lives). Older synthetic assays read-out straight from activation of that first signal from Factor-C. This paper tries to recreate a portion of the cascade before reading out a result. But that now means you not only need to produce the Factor-C, the machinery to ensure Factor-C is properly produced, but you also need at least a few of the downstream components as well. So now you have a multi-component synthetic system - with all the grinding through the search-space and tuning that comes from having to optimize a multi-component biological system in vitro. That's a pretty complicated problem. The paper just came out less then two months ago, and concludes, "The present study supports the future production of recombinant reagents that do not require the use of natural resources". We're on the right track, but it seems this is actually a tricky problem. [1] (Factor-C protein) http://www.uniprot.org/uniprot/P28175 http://www.uniprot.org/uniprot/P28175 [2] (Coagulation Cascade) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/figure/fig1-1753425916681074/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/figure/...

The relevant limulus proteins can be expressed in vitro. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/

jfarlow 9y ago

It looks like (very much like insulin production in vitro) the proteins involved here are highly modified after translation. Some proteins are usable right after they are produced by the ribosome, some require extensive modification to be useful. It looks like Factor-C [1] needs substantial posttranslational by machinery that is not present to the organisms commonly used for industrial protein production. So not only do you have to produce the protein synthetically, but you also have to produce the modification machinery synthetically. An additional challenge is that the readout is at the bottom of a signal-amplified cascade [2]. And that cascade magnifies any error you have at the top (which is where that finicky Factor-C lives). Older synthetic assays read-out straight from activation of that first signal from Factor-C. This paper tries to recreate a portion of the cascade before reading out a result. But that now means you not only need to produce the Factor-C, the machinery to ensure Factor-C is properly produced, but you also need at least a few of the downstream components as well. So now you have a multi-component synthetic system - with all the grinding through the search-space and tuning that comes from having to optimize a multi-component biological system in vitro. That's a pretty complicated problem. The paper just came out less then two months ago, and concludes, "The present study supports the future production of recombinant reagents that do not require the use of natural resources". We're on the right track, but it seems this is actually a tricky problem. [1] (Factor-C protein) http://www.uniprot.org/uniprot/P28175 http://www.uniprot.org/uniprot/P28175 [2] (Coagulation Cascade) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/figure/fig1-1753425916681074/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302069/figure/...