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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

Immunoflouroscence microscopy of L.lactis interacted with the TRP-224-472-cA glycoprotein. (i) DAPI nucleus staining for L. lactis cells (ii) FITC conjugated Secondary Antibody Staining. aL. lactis interacted with cA protein (expressed and purified E.coli), (b) L. lactis interacted with wild type CHO-S cells crude intracellular protein in 1 x PBS, (c) L. lactis interacted with TRP-224-472-cA glycoprotein
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Fig4: Immunoflouroscence microscopy of L.lactis interacted with the TRP-224-472-cA glycoprotein. (i) DAPI nucleus staining for L. lactis cells (ii) FITC conjugated Secondary Antibody Staining. aL. lactis interacted with cA protein (expressed and purified E.coli), (b) L. lactis interacted with wild type CHO-S cells crude intracellular protein in 1 x PBS, (c) L. lactis interacted with TRP-224-472-cA glycoprotein

Mentions: Cell wall anchoring of glycosylated target protein, TRP-224-472-cA was analysed to determine the bio-functionality of cA, the cell wall anchoring LysM motif. It was observed that the L. lactis MG1363 cells treated with TRP-224-472-cA and cA protein alone (positive control for immunofluorescence microscopy) respectively, emitted green fluorescence signals in contrast to those treated with the negative control (L.lactis treated with wild-type CHO-S crude protein in 1 × PBS) (Fig. 4). This confirmed that the target glycoprotein, TRP-224-472-cA, was anchored to the cell wall of L. lactis MG1363. In order to determine if the location of the target glycoprotein correlated with the presence of the L. lactis, DAPI staining was performed. As shown in Fig. 4, TRP-224-472-cA glycoprotein co-localized with L. lactis. Therefore, the target TRP-224-472-cA glycoprotein was expressed in-trans by L. lactis cells through the successful anchoring of the target to its cell wall.Fig. 4


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)

Immunoflouroscence microscopy of L.lactis interacted with the TRP-224-472-cA glycoprotein. (i) DAPI nucleus staining for L. lactis cells (ii) FITC conjugated Secondary Antibody Staining. aL. lactis interacted with cA protein (expressed and purified E.coli), (b) L. lactis interacted with wild type CHO-S cells crude intracellular protein in 1 x PBS, (c) L. lactis interacted with TRP-224-472-cA glycoprotein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Immunoflouroscence microscopy of L.lactis interacted with the TRP-224-472-cA glycoprotein. (i) DAPI nucleus staining for L. lactis cells (ii) FITC conjugated Secondary Antibody Staining. aL. lactis interacted with cA protein (expressed and purified E.coli), (b) L. lactis interacted with wild type CHO-S cells crude intracellular protein in 1 x PBS, (c) L. lactis interacted with TRP-224-472-cA glycoprotein
Mentions: Cell wall anchoring of glycosylated target protein, TRP-224-472-cA was analysed to determine the bio-functionality of cA, the cell wall anchoring LysM motif. It was observed that the L. lactis MG1363 cells treated with TRP-224-472-cA and cA protein alone (positive control for immunofluorescence microscopy) respectively, emitted green fluorescence signals in contrast to those treated with the negative control (L.lactis treated with wild-type CHO-S crude protein in 1 × PBS) (Fig. 4). This confirmed that the target glycoprotein, TRP-224-472-cA, was anchored to the cell wall of L. lactis MG1363. In order to determine if the location of the target glycoprotein correlated with the presence of the L. lactis, DAPI staining was performed. As shown in Fig. 4, TRP-224-472-cA glycoprotein co-localized with L. lactis. Therefore, the target TRP-224-472-cA glycoprotein was expressed in-trans by L. lactis cells through the successful anchoring of the target to its cell wall.Fig. 4

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