Development of CBM-Fusion Protein with Oligomerization Domain (OD) from Pseudomonas aeruginosa Pseudaminidase
Carbohydrate binding module (CBM) from Vibrio cholerae sialidase is located at the N-terminal region which flank the central catalytic domain of the protein. It is belong to the Family 40 which recognizes single sialic acid moiety as the binding ligand. Protein usually tend to have weaker binding...
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| Main Authors: | , , |
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| Format: | Conference or Workshop Item |
| Language: | English English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://eprints.unisza.edu.my/2519/1/FH03-FBIM-19-30933.pdf http://eprints.unisza.edu.my/2519/2/FH03-FBIM-19-30935.pdf http://eprints.unisza.edu.my/2519/ |
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| Summary: | Carbohydrate binding module (CBM) from Vibrio cholerae sialidase is located at the
N-terminal region which flank the central catalytic domain of the protein. It is belong
to the Family 40 which recognizes single sialic acid moiety as the binding ligand.
Protein usually tend to have weaker binding affinity towards its substrate, therefore a
multivalent approach was carried out in order to enhance protein binding affinity via
an avidity effect. In this study, DNA fragment encoding the CBM40 from V. cholerae
sialidase was amplified by PCR method before cloning in E. coli BL21(DE3) using
pET-22(b+) as a vector. The gene composed of 174 amino acids from an open
reading frame comprising 522 bp nucleotide sequence. CBM40-fusion protein was
developed by linking the second domain known as Oligomerization domain (OD)
which isolate from Pseudomonas aeruginosa pseudaminidase. The domain consists of
an open reading frame of 321 bp with the ability to form a trimeric protein molecule.
The OD domain was amplified by PCR method with the addition of BamH1 and EcoR1
sites before ligation was performed on pET-22b(+) vector. In addition, five amino
acids linker length was generated to flank in the CBM and the OD domain. Choices of
fusion linkers were designed composed of flexible residues like glycine (G) and serine
(S) which are flexible group of amino acids. This recombinant fusion construct was
cloned into the pET-22b(+) vector and transform into E. coli BL21 (DE3) host.
Positive recombinant fusion molecule was identified by colony PCR and confirmed by
DNA sequencing techniques. A comprehensive characterization of the recombinant
fusion proteins is currently under evaluation. This CBM-OD construct has a big
potential to be developed as a therapeutics agent to combat harmful pathogens
which recognize sialic acid as the binding substrate. |
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