Difference between revisions of "Part:BBa K4726002"
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===Usage and Biology===
 
===Usage and Biology===
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This encodes a α-galactosidase from <i>Bacteroides fragilis</i> [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 encodes an α-galactosidase from <i>Bacteroides fragilis</i> [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 H antigen. This enzyme has a similar affinity and catalytic activity towards B antigen as 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 antigen, whereas GH110a is only towards branched B antigen. This could prove to be promising for xenotransplantation purposes[1].
+
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.
 
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.

Revision as of 01:15, 10 October 2023


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.


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.