Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 80

The connector protein has been reengineered and inserted into lipid bilayers [ 42 ].
This is the first instance demonstrating the incorporation of a protein that is
neither a membrane protein nor an ion channel into an artificial membrane. The
conductance of each pore in the bilayer lipid membrane (BLM) is almost identical
and is perfectly linear with respect to the applied voltage. Numerous transient
current blockade events in real time induced by dsDNA are consistent with the
dimensions of the channel and of dsDNA. Furthermore, the connector channel is
stable under a wide range of experimental conditions, including high salt and
extreme pH [ 43 ]. The robust properties of the connector nanopore have made it
possible to develop a simple, reproducible approach for pore quantifications
[ 43 ]. More recently, we reported that the phi29 connector channel exhibits a
one-way traffic property for dsDNA translocation [ 44 ]. The most significant
advantage of our system, different from other well-studied systems, is that the
phi29 connector has a larger channel allowing for the passage of both dsDNA
and ssDNA. The larger pore size is also advantageous in that it makes it easier
for channel modifications for the insertion or conjugation of chemical groups for
enhanced sensing applications. These findings have important implications since
artificial membrane architecture for DNA packaging motor would allow detailed
investigations into discrete mechanisms of motor operation as well as future
avenues for therapeutic dsDNA packaging, sampling, and delivery.
4.2 Reengineering, Expression and Purification
of the Phi29 Connector
4.2.1 Reengineering of Phi29 Connector
The construction of the plasmid for the expression of the connector protein and the
assembly of the dodecameric connector has been reported previously [ 41 ].
The subsequent terminal modifications of the connectors have also been described
[ 40 , 45 , 46 ]. Briefly, the modification of one of the plasmids was, for example,
achieved by a two-step polymerase chain reaction (PCR). First, the primer pair
F1-R1 was used to amplify the GP10 gene. The first PCR product was used as a
template for a second step PCR with primer pair F1 and R2, which contained affinity
Tags (His-Tag or Strep-Tag) as well as the restriction sites for NdeI and XhoI,
respectively. The second PCR product was digested with NdeI/XhoI and ligated into
the NdeI/XhoI sites of the vector pET-21a(+) (Novagen) to generate the plasmid.
4.2.2 Expression and Purification of the Connector
Plasmid pETgp10-Cstrep or -Chis was transformed into the Escherichia coli strain
HMS174 (DE3) for protein expression. A volume of 10 mL of the E. coli culture