textThe twin-arginine translocation (Tat) pathway of Escherichia coli provides a
novel method for the export of proteins from the cytoplasm to the periplasm.
Remarkably, it allows for large, folded proteins to cross the inner membrane, with no
apparent effect on cell viability. Protein export from the cytoplasm is employed in a
variety of biotechnological applications including manufacturing and protein engineering.
However, until the discovery of the Tat pathway, such applications relied on the Sec
pathway, in which proteins transverse the lipid bilayer membrane in an extended form
and protein folding takes place after export. Many proteins of biotechnological interest
are not compatible with export via the “classical” Sec pathway. Thus, the export of such
Sec-incompatible proteins via the Tat pathway could open the way for new biotechnology
applications.
This work explores several mechanistic, physiological and technology-related
aspects of Tat export In all organisms, proteins are secreted by virtue of a peptide
extension, or signal peptide, comprising 15-45 amino acids. The signal peptide serves as
a “zip code” and is cleaved after export. E. coli contains 29 putative Tat-specific signal
peptides but their ability to mediate export via the Tat pathway has not been confirmed
experimentally. The export pathway (Tat or Sec) utilized by this set of 29 signal peptides
was characterized using fusions to protein reporters. The reporter proteins chosen for this
study are functional only when translocated across the membrane either via the Sec or Tat
pathways. Surprisingly, it was found that while 11/29 signal peptides are Tat-specific
and 2/29 are Sec-specific, a set of 16/29 signal peptides were able to direct export via
both the Tat and Sec pathways. Interestingly, increasing the charge of the region
surrounding the cleavage site – particularly the N-terminus of the mature protein –
resulted in Tat specificity.
In separate studies we showed that in addition to an appropriate signal sequence,
proteins destined for export via the Tat pathway must complete their folding in the
cytoplasm. Partially folded proteins are not competent for export via this pathway. The
requirement for folding in the cytoplasm prior to export was demonstrated by using an
elegant system whereby the conformation of the polypeptide chain in the cytoplasm could
be controlled using conditions that supported or prevented the formation of disulfide
bonds. These results led us to propose that the Tat pathway contains an intrinsic folding
quality control mechanism, a concept that has since been widely adopted in the literature.
Finally, a new methodology was developed for the engineering of proteins that
require the cytoplasmic machinery to fold but must then be exported into the bacterial
periplasmic space. Specifically, we created a Tat-based system to enable the display of
proteins on filamentous phage, a prerequisite for the high-throughput screening of protein
libraries. This system relies on the forced dimerization of the phage coat protein p3,
which is exported into the periplasm via the Sec pathway, and the protein that is to be
displayed, which is exported via the Tat pathway. Forced dimerization of p3 and the
desired protein in the periplasm was mediated by coiled-coil interactions. We further
demonstrated that this Tat-dependent display platform shows promise for use in
engineering ligand-binding loops into green fluorescent protein (GFP) for sensor
applications.Chemical Engineerin
