Part:BBa_K1352012

Antigen 43 (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and 488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non-covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60oC. Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence. Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in E.coli biofilm formation. Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kjærgaard et. al., 2002).

This BioBrick is a modified version of the Bba_K1352001 BioBrick. It is composed of an Ag43 coding sequence with an in-frame FLAG epitope tag flanked by BglII and HindIII multiple cloning sites inserted within the alpha-subunit in front of codon 148 of Ag43. Expression of Ag43 is under the control of the pBAD promoter; it includes a ribosome binding site as well as two transcriptional terminators.Two 17 amino acids long beta-hairpins were removed from the Ag43 coding sequence in order to remove aggregation properties.

The shape of α-subunit of Ag43 resembles letter L. It consists of a 'β-helix domain [which form] the stem of the letter L, followed by three rungs flanked by four β-hairpin motifs that bend the protein by about 110° and a C-terminal (...) parallel β-helix domain [which forms] the bottom of the letter L' (Heras et. al., 2014). It has been indicated that this unique shape plays a crucial role in cell-to-cell aggregation via velcro-like mechanism, in which α-subunits form a dimer by coling around each other. This interaction is strengthened by Van der Waals interactions, hydrogen bonds and salt bridges facilitated by the L-shape (Heras et. al., 2014). Recent research demonstrates that disruption of the bend and straightening of the shape by removal of two β-hairpin sequences eliminates self-association of Ag43 proteins (Heras et. al., 2014). Removal of β-hairpins does not interfere with protein translocation to the cell surface membrane.

Hairpin 1 sequence 268 AATVTGTNRLGAFSVVA 284

Hairpin 2 sequence 341 GAAVSGTRSDGKAFSIG 357

Structure of the BioBrick was designed to allow easy insertion of foreign protein sequences of choice at codon 148 with a simple restriction digest with BglII and HindIII followed by ligation. Potential uses of this BioBrick include surface display of foreign peptide sequences when aggregation properties are not desirable and synthetic vaccines production.