Part:BBa_K4726002

α-galactosidase GH110b_Bacteroides fragilis

Usage and Biology

This encodes an α-galactosidase from Bacteroides fragilis [1]. This enzyme is available on Uniprot with the entry Q5LGZ8. We added a 6X his-tag on the C-terminus of the protein.

This enzyme removes the α-galactosyl moiety leading to the H antigen. This enzyme has a similar affinity and catalytic activity towards B antigen to the previously used α-galactosidase (GH110a) from the same bacteria (BBa_K3717006) [1]. However, GH110b was shown to be active towards both branched and linear B antigens, whereas GH110a is only active towards branched B antigen. This could prove to be promising for xenotransplantation purposes[1].

The expression of this enzyme led to significant proteotoxicity shown by an impaired growth of Escherichia coli BL21 (DE3). We thus recommended to use Escherichia coli Rosetta (DE3) pLysS which helps stabilizing pET recombinants encoding proteins.

Characterization and results

The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #9, we see overproduction of the aforementioned protein (67.834 KDa).

Figure 1.SDS-PAGE electrophoresis of purification steps.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1213
  Illegal EcoRI site found at 1714
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1213
  Illegal EcoRI site found at 1714
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1213
  Illegal EcoRI site found at 1714
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1213
  Illegal EcoRI site found at 1714
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1213
  Illegal EcoRI site found at 1714
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Liu QP, Yuan H, Bennett EP, Levery SB, Nudelman E, Spence J, Pietz G, Saunders K, White T, Olsson ML, Henrissat B, Sulzenbacher G, Clausen H. Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen. J Biol Chem. 2008 Mar 28;283(13):8545-54. doi: 10.1074/jbc.M709020200. Epub 2008 Jan 28. PMID: 18227066; PMCID: PMC2417185.