As can be seen from the protein expression SDS-PAGE, although much of the cell lysis on the gel impairs any discernment of exact protein expression, there appears to be an overexpression band at around the 35 kDa mark. This is likely evidence of the expression of our encapsulin monomer under reducing conditions. To assess the possibility of cellular expression of encapsulins, future non-reducing Native-PAGE gels are being conducted to visualize the assembling capacity of the nanocompartment.
Future directions
Future directions for our project will likely focus on producing assembled encapsulin nanocages containing both natural and engineered metal binding proteins at large scales for biochemical and structural characterization. An open question in encapsulin research is the efficiency of which heterologous cargo can be targeted to the interior of the protein nanocage, which may have implications for the efficiency of our system. To assess the degree to which our metal-binding proteins are encapsulated, negative stain transmission electron microscopy or cryogenic electron microscopy of purified nanocages could be used. In future designs, the sequence of the targeting peptide appended to the cargo proteins may also be varied to or from the consensus sequence to manipulate the degree of cargo internalization accordingly.
While preliminary results suggest that metal-binding proteins produced engineered using ProteinMPNN may have improved soluble expression compared to the wild type, it is uncertain as to whether they retain their native metal binding function. To ascertain this, we can employ differential scanning fluorimetry (DSF) to gain information about the kinetics of metal binding. An alternative approach that could make use of the advanced joint mass spectrometry facilities at the ANU is native mass spectrometry, which may allow us to better resolve the metal binding dynamics of our engineered variants in their native state. This method may also have greater utility for evaluating the binding capabilities of these proteins once internalized within the encapsulin.