Scientists often tackle similar problems, but it’s uncommon for them to independently find solutions to the same problem at the same time.
That’s what happened with two scientific teams that incorporated an important biological process called tyrosine phosphorylation into genetically modified bacteria.
Their two studies on making phosphotyrosine in genetically modified E. coli were published Monday. Their work may lead to greater understanding of proteins incorporating this biomolecule, and dysfunctions of this process, including cancer.
The coincidences don’t stop there. Many of these scientists work almost next door to each other in La Jolla, and know each other well.
And they didn’t know they were publishing in the same journal, Nature Chemical Biology.
---The other included a scientist from the Salk Institute for Biological Studies, Tony Hunter, who pioneered the field of tyrosine phosphorylation research nearly four decades ago. This study can be found at j.mp/salkphos.
Lei Wang, that study’s senior author, recently moved from the Salk Institute to the University of California San Francisco, the affiliation given on the study.
Hunter is famous for discovering a class of enzymes called tyrosine kinases that perform tyrosine phosphorylation. At last count 90 genes are known to code for these enzymes. Dozens of cancer therapies have arisen from this discovery, and more are in testing.
Hunter said he didn’t know of the TSRI-led study, which I supplied to the Salk Institute for comment. (I likewise supplied the Salk Institute study to TSRI, and will report back if that team comments).
“The irony is that Lei Wang, the senior author of the paper I’m on, was a graduate student with Pete Schultz,” Hunter said.
Studying tyrosine phosphorylation is convoluted, because the genetic mechanism for making phosphotyrosine is indirect.
Proteins incorporating phosphotyrosine are made post-translationally, by attaching a phosphate group to tyrosine. So the genetic code for the original and phosphorylated forms of a protein is identical. Moreover, phosphorylated proteins can be de-phosphorylated.
While genetics at some level obviously determines how post-translational modifications occur, it’s not nearly as neat and simple as directly transcribing a sequence at one end and churning out a protein at the other end.
Both teams pursued the idea of directly making phosphotyrosine by tweaking the genetic code of E. coli, the ubiquitous workhorse lab bacterium. The result is an expanded genetic code.
Such tweaking is possible because the genetic code is degenerate. A codon that specifies the same thing as another codon is redundant. It can be switched to specify another amino acid, one not among the 20 naturally encoded ones.
This approach of incorporating “unnatural” amino acids into proteins made in E. coli was pioneered by Schultz’s group at TSRI, making this an obvious approach to use to install phosphotyrosine in proteins, Hunter said.
However, there are major hurdles in actually getting E. coli to produce phosphotyrosine- containing proteins. One is that phosphotyrosine doesn’t readily penetrate the cell wall of E. coli, because they both are negatively charged, and so repel each other.
Analogs of phosphotyrosine have been developed, but they have drawbacks that prevent general use.
“The two groups have solved this in a different way,” Hunter said.
The Scripps Research Institute-led team invented a “clever trick,” Hunter said, to increase cellular uptake of phosphotyrosine and an analog. These were attached to a second normal amino that allows them to be taken up by a transporter protein. After gaining entry inside the cell, the complex is cleaved, freeing phosphotyrosine and the analog again.
The next step was to place both the phosphotyrosine and the analog at specific sites in recombinant proteins, by swapping out a codon. That used a method Schultz and Wang developed in 2001 at TSRI, Hunter said.
The result was a good protein yield in E. coli.
“Finally, we are attempting to adapt a similar strategy for eukaryotic cells to allow the controlled expression of tyrosine-phosphorylated proteins in real time,” the study said.
The study led by Wang that Hunter participated in took a different route to the same end. It made a workable phosphotyrosine analog system that yielded actual phosphotyrosine-containing proteins.
The analog used in the study is stable and neutral, allowing passage through the cell wall. After the protein is made, it’s subjected to a mildly acid solution. This converts the analog into phosphotyrosine at the desired places on the protein.
“Finding the right analog that would get into cells sufficiently was the key,” Hunter said. “And then it’s fairly routine after that.”
A weakness is that the protein must be able to withstand the acid and refold back into its functional state after the acid is gone, Hunter said, but the Wang group successfully made three different phosphotyrosine proteins this way.
The next step is to reproduce this process in eukaryotic cells, preferably mammalian cells, he said.
The Scripps work has been shown to work definitively for just one protein, Hunter said, but it should work for other proteins as well.
Taken together, Hunter said the studies represent an advance to the “Holy Grail” of making phosphotyrosine-containing proteins on demand.
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