Team:Aberdeen Scotland/Parts


Revision as of 23:05, 17 October 2014 by Martynasroka (Talk | contribs)

Team:Aberdeen Scotland/Parts -

Background to Parts Design

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).

Fig.1    Graphic representation of Ag43 autotransporter structure and process of autotransportation.
Source: Kjærgaard et. al., 2000; Van der Woude & Henderson, 2008 (modified).

The shape of α-subunit of Ag43 resembles letter L (Fig.2). It consists of a 'β-helix domain [which forms] 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

Fig.2   A) Graphic representation of Ag43 alpha-subunit; B) Interaction between two alpha-subunits in a velcro-like mechanism of auto -aggregation; C) Disruption of L-shape and linearization of the alpha-subunit eliminates auto-aggregation properties.

Ice Nucleation Protein (INP) is used by bacteria in nature to nucleate ice crystals at slightly sub-zero temperatures; these crystals cause frost-damage to plant tissues which releases nutrients allowing the bacteria to metabolise them. It is another autotransporter and is extremely similar to Ag43. It also has an alpha and beta region and inserts itself into the cell’s outer membrane in basically the same way indicated in the first diagram above. Unlike Ag43 where the foreign proteins are inserted at codon 148, in INP the protein is inserted on the C terminus. INP has been used by a number of researchers and iGEM teams to surface- display proteins of interest. Ag43 is perhaps a better surface-displayer as it protrudes further from the cell surface than INP, but INP is easier to engineer as it has a larger carrying-capacity and the gene is much smaller.