2017 Onwards and Upwards
Lab Note 14
Maria Chavez with Anthony Di Franco and Catherine Soneda
It has been over a year since we launched the Open Insulin Project on Experiment.com. Not only has this year brought us closer to our goals - it’s highlighted to us the impact of insulin monopolies on society and welfare. Despite being a non-profit, volunteer, ever-in-flux community of individuals who are strange and started off as strangers to each other, we’ve bonded not only through our original interests, but our belief that together we can make a statement. We’ve connected with like-minded communities from New York, to Colorado, to the Bay Area; including groups such as The 100 Campaign, Antonio Lamb/Microsynbiotx, and more . We have overcome sub-optimal lab working conditions and faulty equipment, and now can feel confident we will be wrapping up the first phase of the Open Insulin Project.
Meanwhile we’ve seen national media coverage of the ongoing, unexplained and apparently arbitrary price increases for many vital drugs, including insulin. We continue to be motivated by the need for a less expensive insulin option.
As you may recall, the approach we are taking to make insulin is to first produce a green fluorescent protein (GFP) - human proinsulin fusion protein. The GFP makes it easy to visually detect protein expression, and helps in purification by making the whole protein more soluble, while the human proinsulin is what we need to cut and fold into insulin. We recently grew and purified large quantities of our GFP-proinsulin fusion protein. Now we need to digest this with enterokinase to split the GFP from the proinsulin, and then run protein gels to determine whether we have successfully produced human proinsulin, which by itself doesn’t glow or otherwise indicate its presence directly. Once they are split up, each will show up in its own band in the protein gel. By comparing the position of each band with the bands in a size standard, we will determine the sizes of the products of digestion with enterokinase and see if they are consistent with those of GFP and proinsulin.
We ordered enterokinase from a vendor who referred our order to an Israeli supplier, and consequently it was trapped in customs for a while, delaying us. Once it came in we tried a digestion according to some rough guidelines on reactant concentration reaction time and temperature and ran a gel but it was not successful. So we decided to quantify our protein concentration so as to be better able to determine the conditions for the digestion. In order to quantify the concentration, we purchased a BCA kit (a protein quantification assay) and also got lucky and found in auction a UV spectrophotometer for the lab in December, either of which will let us go forward. We’re working on the quantification now and are planning to resume with the digestion within the week.
Currently, Noel can do rigid modeling and rotation of dihedral angles reliably in his simulation software. He has taken a break from the basics of manipulating bond geometry and is looking at efficient ways of searching the conformation space, a higher-level problem where one tests different protein shapes to see which are the lowest in energy and thus the most physically likely. As part of this he downloaded the entire protein databank to play with. He is also looking at an energy function based on the Still equation and Coulomb charge energies to guide the search.
This will support ongoing work on simulating the flexible linker which are key to a strategy Patrik suggested at the beginning of the project as a possible way to guide the assembly of insulin from its A and B chains expressed separately with leucine zippers attached. Leucine zippers are motifs found in nature that attract each other and can bring other parts of a protein together for disulfide bonding. There are rules of thumb about linkers to refer to based in natural linkers found in proteins that have had their structures solved. The simulation is still searching a space of rigid configurations, which is a problem because it leads to spurious clashes of substructures.
Thanks for your support!
We look forward to working on making Open Insulin a reality in 2017 and continuing to push for generic and less expensive sources of medications. We couldn’t have started our work without your support, and we continue to count on it. Supply, reagent, and equipment purchases and renovations to the lab at CCL to bring things up to code, install plumbing and power for our new equipment have put pressure on our budget. Please consider donating to the Open Insulin project and CCL to keep our work on track!