By Erin Shanahan

In spring 2014, Dr. Paul Smith, from Fordham’s Department of Chemistry, got a phone call regarding a mysterious protein referred to as E4orf1. Most properties of the protein were unknown at the time he got the call. However, currently, Dr. Smith’s team is making headway towards learning more about the biomolecule.

Specifically, Dr. Smith is looking to create a high-resolution x-ray crystal structure of the protein as well as detail some of the protein’s biophysical characteristics. This task has proven to be very difficult, especially considering molecules are invisible to the naked eye.

The scientific community became interested in this protein after the Rafii lab in Howard Hughes Medical Institute observed the effects of expressing a single adenovirus gene on endothelial cells. The lab found that the protein E4orf1 may allow for the better growth of human umbilical vein endothelial cells (or PECs).

PECs act as a laboratory model system. These PECs are used for the study of the function and pathology of endothelial cells.
Prior to this finding regarding E4orf1, PECs were problematic for multiple reasons.

First, the creation of PECs require many growth factors, specific serums, and other complicated cellular extracts, such as cow brains. These growth agents are problematic because the human immune system will reject them. Therefore, this growing concoction is not useful for humans. In addition, growing PECs in this way creates immortal cell lines, which look more like cancer cells than healthy human PECs found in the body. Finally, these PECs are tricky to grow.

However, the Rafii lab in Howard Hughes Medical Institute found that PEC’s expressing the E4orf1 protein can be grown in serum. In other words, the E4orf1 protein nullifies the nasty cow-brain growing concoction issue with PEC’s.

In addition, the Rafii lab found that PEC’s expressing the E4orf1 protein can display “normal” angiogenic properties. In other words, PEC’s expressing the E4orf1 protein does not create immortal cell lines, but instead creates cells that display normal cell growth and death.

Although the Rafii lab had found some interesting information about the E4orf1 protein in relation to PEC’s, much about the protein remains unknown.

Specifically, back in spring 2014 no lab had established purification of the protein’s structure. In addition, the enzymatic properties were unknown. Finally, structure and function relationships, or mutations of the protein, were also unknown.
This is where Dr. Smith and his team of research assistants at Fordham came into play.

Dr. Smith received a phone call regarding this protein and its unknown structure and was asked to research its molecular properties. Specifically, his lab is looking to create a high-resolution x-ray crystal structure. Collateral goals include finding functional and biophysical characteristics.

Dr. Smith’s lab was the first to express and purify many milligrams of E4orf1. This proliferation of pure E4of1 was essential in order to run further tests on the protein.

Although Dr. Smith has not created a high-resolution x-ray crystal structure yet, his lab is still very hopeful.

“Crystallization is a pretty frustrating and time-consuming,” Dr. Smith said. “It involves mixing highly concentrated protein with a cocktail of chemicals that alter ionic strength, osmolarity, and pH so as to promote gentile precipitation into a lattice. It’s not uncommon for thousands of trials to be necessary.”

To help with the process, Dr. Smith has programed a robot to do the majority of the concocting. Currently over 5,000 trials have been done, but still no crystal.

Besides working on crystallization, Dr. Smith’s lab has run several other tests on the protein as well. Most recently, the lab found that E4 has very strong aggravating properties. In other words, when the protein reacts in a specific chemical reaction, it gives off a very strong, cement-like discharge.

“I’ve never seen a protein precipitate so strongly. I would sell it for glue if I could,” he joked.