HaloTag

Site-specific copy number variations in cancer: A story begins to unfold

Posted on by Michele Arduengo, PhD
Designed by Nick Klein for ISO-form, courtesy of Promega.
Designed by Nick Klein for ISO-form, courtesy of Promega.

Tumor cells are characterized by many features: including uncontrolled proliferation, to loss of contact inhibition, acquired chromosomal instability and gene copy number changes among them. Some of those copy number changes are site-specific, but very little is known about the mechanisms or proteins involved in creating site-specific copy number changes. In a recently published Cell paper, Black and colleagues, propose a mechanism for site-specific copy number variations involving histone methylation proteins and replication complexes.

Previous work from Klang et al. had shown that local amplification of chromosomal regions occurs during S phase and that chromatin structure plays a critical role in this amplification (2), and other work by Black and colleagues (3) implicated KDM4A in changing timing of replication by altering chromatin accessibility in specific regions. Other research also had shown that KDM4A protein levels influence replication initiation and that KDM4A has a role in some DNA damage response pathways (4,5).  Looking at the body of work, Black et al. hypothesized that KDM4A, with its roles in replication, might possibly provide link into the mechanism of site-specific copy number variation in cancer. Continue reading “Site-specific copy number variations in cancer: A story begins to unfold”

Another Step Closer to Understanding Epigenetic Gene Regulation

Posted on by Terri Sundquist
Chromatin fiber

Back when I was a graduate student (more than a few years ago), I remember hearing another student joke that if a member of his thesis committee asked him to explain an unexpected or unusual result, he was going to “blame” epigenetics. At that time, the study of epigenetic gene regulation was in its infancy, and scientists had much to learn about this mysterious regulatory process. Fast forward to today, and you’ll find that scientists know a lot more about basic epigenetic mechanisms, although there is still plenty to learn as scientists discover that the topic is much more complicated than initially thought, as is often the case in science. A recent EMBO Journal article is contributing to our knowledge by shedding light on the role of the TET family of DNA-modifying enzymes in epigenetics (1).

Continue reading “Another Step Closer to Understanding Epigenetic Gene Regulation”

Screening for Protein Activity Using Cell-Free Expression

Posted on by Gary Kobs

The analysis of functional protein typically requires lengthy laborious cell based protein expression that can be complicated by the lack of stability or solubility of the purified protein. Cell free protein expression eliminates the requirement for cell culture thus providing quick access to the protein of interest (1).

The HaloTag® Technology provides efficient, covalent and oriented protein immobilization of the fusion protein to solid surfaces (2).

Leippe et al. demonstrated the feasibility of using cell free expression and the HaloTag technology to express and capture a fusion protein for the rapid screening of protein kinase activity (3). The catalytic subunit of human cAMP dependent protein kinase was expressed in a variety of cell free expression formats as a HaloTag fusion protein. The immobilized cPKA fusion protein was assayed directly on magnetic beads in the active form and was shown to be inhibited by known PKA inhibitory compounds.

Therefore this unique combination of protein expression and capture technologies can greatly facilitate the process of activity screening and characterization of potential inhibitors

References

  1. Zhao, K.Q. et al. (2007) Functional protein expression from a DNA based wheat germ cell-free system. J. Struc. Funct. Genomics. 8, 199-208.
  2. Los, G.V. and Wood, K. (2007) The HaloTag: A novel technology for cell imaging and protein analysis. Meth. Mol. Biol. 356, 195-208
  3. Leippe DM, Zhao KQ, Hsiao K, & Slater MR (2010). Cell-free expression of protein kinase a for rapid activity assays. Analytical chemistry insights, 5, 25-36 PMID: 20520741