Team:Hawaii/Demonstrate
DEMONSTRATE
SUMMARY
DEMONSTRATION OF VLP FORMATION FROM DETERMINED CLEAVAGE SITE
We have confirmed virus-like particle (VLP) formation from expression of our experimentally determined Gag construct through electron microscopy (EM). This construct was designed after our determination of the protease cleavage site through a protease assay and bioinformatic putative protease cleavage site predictions.
This is the first time anyone has observed the particles from the centromeric retrotransposon (CR) element of the CR2 subfamily in Zea mays. We have summarized our steps taken to isolate the protease cleavage site for VLP formation and show our EM results below. More details can be found on our Experiments page.
CREATION OF THE GAG-PROTEASE SEQUENCE
Consensus sequences of the centromeric retrotransposon subfamily 2 of Zea mays were used to create our first construct spanning from the beginning of the Gag to the beginning of the reverse transcriptase domain. This allows us to maximize the residues in which the protease may cleave to form mature virus-like particle (VLP) proteins.
AUTOCATALYTIC PROTEASE ACTIVITY AND MASS SPEC REVEAL CLEAVAGE SITE
Upon expression of the construct in BL21 cells, autocatalytic activity is observed and 3 bands are seen representing partial degradation fragments after protease cleavage. These bands are sent to the Taplin Mass Spectrometry facility and tryptic fragments reveal a protease cleavage site downstream of the nucleocapsid. This is used to design our 2nd construct for expression.
VIRUS-LIKE PARTICLES OBSERVED AFTER VLP ASSEMBLY
Proteins are purified after induction and subjected to various VLP assembly conditions. Analysis of proteins under the EM reveal virus-like particles. Constructs reflecting other possible cleavage sites are expressed and analyzed. The construct with the experimentally determined cleavage site appears to be more spherical and complete. This demonstrates that our construct does allow for VLP formation.