Part:BBa_K1978000

TorA-BtuF

The TorA-BtuF Biobrick consists of a TorA signal sequence linked to BtuF, a protein capable of binding vitamin B₁₂. The TorA signal peptide allows export of fully-folded proteins through the inner membrane via the Tat (Twin-Arginine translocation) system. This construct thus enables export of vitamin B₁₂ bound to BtuF out of the cytoplasm. The TorA sequence codes for an amino-terminal signal peptide that harbours a twin-arginine motif which is vital for the recognition by the Tat system. The TorA signal sequence and the sequence coding for BtuF are connected by a linker of 15 bases, coding for the five amino acids following the signal peptide in trimethylamine-N-oxide reductase from E.coli. Moreover, an AxA motif is present, which leads to cleavage by the leader peptidase

Usage and Biology

BtuF

BtuF is the periplasmic binding protein for the vitamin B12 transporter BtuCD from E.coli (Cadieux et al., 2002). While some bacteria and archaea are capable of its synthesis, E.coli belongs to the majority of prokaryotes that contain transport systems to import B₁₂ (Warren et al., 2002). Its transmembrane transport is achieved by the Btu (B twelve uptake) system composed of BtuB, an outer membrane TonB-dependent transporter (Cadieux et al., 1999), and the ABC transporter BtuCDF, which is located in the inner membrane. While BtuC and BtuD compose respectively the trans-membrane domain and the ABC (Bassford et al., 1977), BtuF is the periplasmic binding protein. It has a size of 30.19 kDa and is composed of two globular domains, between which vitamin B₁₂ is bound, linked by a rigid interdomain α-helix (Karpowich et al., 2003). A crystal structure of the protein bound to B12 is available (PDB1N4A; Karpowich et al., 2003).

The Tat export pathway and its signal peptide

In bacteria and archaea, proteins located outside the cytoplasm can reach their destination via either the Sec or the Tat (twin-arginine translocation) export pathway. While the Sec system translocates proteins in an unstructured state, the Tat apparatus has the unusual property of transporting fully folded proteins (Palmer and Berks, 2012). This system is very flexible in regard to the types of proteins that can be exported and the number of exported proteins highly differs between organisms. The E.coli Tat system is capable of transporting substrates up to 70 Å in diameter (Berks et al., 2000). Many exported proteins containing non-covalently bound cofactors use this pathway, because the cofactor is held in place by the protein folding. The Tat pathway is only used by proteins containing certain types of cofactors that are classified as metal-sulphur clusters or nucleotide based cofactors, which include among others also cobalamins (Berks et al., 2003).

Proteins are targeted to the Tat apparatus by amino-terminal signal peptides that are normally cleaved by an externally facing signal peptidase (Lüke et al., 2009). The overall architecture is similar to Sec signal peptides and includes a tripartite structure with a basic n region at the N terminus, a hydrophobic h region in the middle and a polar c region at the C terminus. The key element of a Tat signal peptide is the highly conserved twin-arginine motif, defined as SRRXFLK. The two arginines are almost always invariant, while the other residues occur with a frequency of > 50 %. The amino acid at position X is usually polar (Palmer and Berks, 2012).

The TorA signal peptide used in this biobrick belongs to the trimethylamine-N-oxide reductase from E. coli, a well characterized protein exported via the Tat system. Using this signal peptide, it has already been achieved to export a heterologous protein normally transported by the Sec pathway through the Tat system (Christóbal et al., 1999).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]