Part:BBa_K257001

Gene 3 protein of filamentous phages without the 18 first amino acids

Gene 3 protein (g3p) without signal peptide. Adding a site SacI for inframe fusion.

Usage and Biology

• Infection of Escherichia coli by filamentous bacteriophages as M13, fd, f1, is mediated by the phage gene 3 protein (g3p or pIII). This protein of 406 amino acid residues, has a signal peptide, two N-terminal domains and one C-terminal domain, separated by two flexible glycin-rich linkers. All three domains are indispensable for phage infectivity.

• The signal peptide (1-18aa) address the protein to the cell membrane before being cleaved. (We deleted it).
• The first N-terminal domain (N1) binds to the bacterial periplasmatic domain of TolA (TolAII), receptor presumably at the inner face of the outer membrane.
• The second N-terminal domain (N2) gives recognition of the host cell by binding the F-pilus on the surface of E. coli. F-pilus is encode by the F episome of male E. coli, and is the primary receptor of the host cell.
• In fact, N1 and N2 interact with each other to form a blocked di-domain (N1G1N2). The binding of N2 to the tip of the bacterial F-pilus releases N1, which becomes free to interact with its receptor TolA (TolAIII).
• The C terminus (CT) of g3p anchors the g3p in the phage coat by interacting with phage coat protein 6, at the tip of the phage. Its seem that phages are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of g3p.
• N1, N2 and N3 domain are linked by flexible glycin-rich domains (G1 and G2). G1 is composed of four tandem copies of the sequence Glu-Gly-Gly-Gly-Ser. In a recent study it has been showed that it may have an active role in F-pilus-dependent infection.
• Fusion of peptides or proteins to the N-terminus of intact g3p does not compromise infectivity of the phage, but insertion of polypeptides between N2 and N3 appear to reduce the infectivity.

NB: The GC rich linker domain between residue 240 and 260 is made of repetition of the same motif. This sequence contain an extra repetition with respect to most sequences deposited in public database. However this should not affect the function of the protein.

Reference

See IGEM Paris 2009 team project : http://2009.igem.org/Team:Paris/Addressing_overview#A._G3P

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 592
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]