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Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis.

Kalyanasundram J, Chia SL, Song AA, Raha AR, Young HA, Yusoff K - BMC Biotechnol. (2015)

Bottom Line: This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation.The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall.Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia. gs31997@mutiara.upm.edu.my.

ABSTRACT

Background: The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.

Results: A total amount of 33 μg of partially purified TRP-2-cA from ~6.0 g in wet weight of CHO-S cells was purified by His-tag affinity chromatography. The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall.

Conclusions: Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

Show MeSH

Related in: MedlinePlus

Deglycosylation of TRP-224-472-cA fusion protein by PNGase treatment. Lane M: PageRuler™ Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1 : Undigested TRP-224-472-cA fusion protein; Lane 2: Replicate of undigested TRP-224-472 -cA fusion protein Lane 3: PNGase digested TRP-224-472-cA fusion protein; Lane 4: Replicate of PNGase digested TRP-224-472-cA fusion protein
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Fig2: Deglycosylation of TRP-224-472-cA fusion protein by PNGase treatment. Lane M: PageRuler™ Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1 : Undigested TRP-224-472-cA fusion protein; Lane 2: Replicate of undigested TRP-224-472 -cA fusion protein Lane 3: PNGase digested TRP-224-472-cA fusion protein; Lane 4: Replicate of PNGase digested TRP-224-472-cA fusion protein

Mentions: The production of TRP-224-472-cA fusion glycoprotein by pcDNA: trp1-472-cA transfected CHO-S cells was analysed by SDS-PAGE and Western Blotting. Based on the Western Blot, the harvested crude protein supernatant showed TRP-224-472-cA as a specific band detected at ~ 90 kDa (Fig. 2). Since the predicted molecular weight with the absence of glycosylation for TRP-224-472-cA fusion protein was ~ 74 kDa, the detection of a specific band with an excess of ~16 kDa was a preliminary indicator of potential glycan moieties present in the target protein. This was confirmed by PNGase F digestion which resulted in significant reduction in TRP-224-472-cA protein molecular weight from ~90 kDa to ~70 kDa after digestion (Fig. 2). Subsequently, the bio-functionality of the TRP-2 subunit in the target fusion protein was analysed through its ability to catalyse dopachrome tautomerization. The distinct decolorization of the L-dopachrome substrate (reduction in A475 nm) was observed after 20 min incubation at 30 °C in the crude protein fraction containing TRP-224-472-cA fusion protein-substrate mixture, in contrast to wild-type CHO-S cell crude protein-substrate mixture (Table 1). This indicates that the TRP-2 subunit in fusion glycoprotein is enzymatically active.Fig. 2


Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis.

Kalyanasundram J, Chia SL, Song AA, Raha AR, Young HA, Yusoff K - BMC Biotechnol. (2015)

Deglycosylation of TRP-224-472-cA fusion protein by PNGase treatment. Lane M: PageRuler™ Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1 : Undigested TRP-224-472-cA fusion protein; Lane 2: Replicate of undigested TRP-224-472 -cA fusion protein Lane 3: PNGase digested TRP-224-472-cA fusion protein; Lane 4: Replicate of PNGase digested TRP-224-472-cA fusion protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696278&req=5

Fig2: Deglycosylation of TRP-224-472-cA fusion protein by PNGase treatment. Lane M: PageRuler™ Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1 : Undigested TRP-224-472-cA fusion protein; Lane 2: Replicate of undigested TRP-224-472 -cA fusion protein Lane 3: PNGase digested TRP-224-472-cA fusion protein; Lane 4: Replicate of PNGase digested TRP-224-472-cA fusion protein
Mentions: The production of TRP-224-472-cA fusion glycoprotein by pcDNA: trp1-472-cA transfected CHO-S cells was analysed by SDS-PAGE and Western Blotting. Based on the Western Blot, the harvested crude protein supernatant showed TRP-224-472-cA as a specific band detected at ~ 90 kDa (Fig. 2). Since the predicted molecular weight with the absence of glycosylation for TRP-224-472-cA fusion protein was ~ 74 kDa, the detection of a specific band with an excess of ~16 kDa was a preliminary indicator of potential glycan moieties present in the target protein. This was confirmed by PNGase F digestion which resulted in significant reduction in TRP-224-472-cA protein molecular weight from ~90 kDa to ~70 kDa after digestion (Fig. 2). Subsequently, the bio-functionality of the TRP-2 subunit in the target fusion protein was analysed through its ability to catalyse dopachrome tautomerization. The distinct decolorization of the L-dopachrome substrate (reduction in A475 nm) was observed after 20 min incubation at 30 °C in the crude protein fraction containing TRP-224-472-cA fusion protein-substrate mixture, in contrast to wild-type CHO-S cell crude protein-substrate mixture (Table 1). This indicates that the TRP-2 subunit in fusion glycoprotein is enzymatically active.Fig. 2

Bottom Line: This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation.The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall.Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia. gs31997@mutiara.upm.edu.my.

ABSTRACT

Background: The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.

Results: A total amount of 33 μg of partially purified TRP-2-cA from ~6.0 g in wet weight of CHO-S cells was purified by His-tag affinity chromatography. The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall.

Conclusions: Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

Show MeSH
Related in: MedlinePlus