Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2

Food and commensal lactic acid bacteria (LAB) surface display system exploitation for bacterial, viral, or protozoal antigen delivery has received immense interest currently. The Generally Regarded as Safe (GRAS) status of LAB such as Lactococcus lactis coupled with non-recombinant strategy of in-...

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Main Author:	Kalyanasundram, Jeevanathan
Format:	Thesis
Language:	English
Published:	2015
Subjects:	Glycosylation Therapeutics - Research
Online Access:	http://psasir.upm.edu.my/id/eprint/60443/1/FBSB%202015%2013IR.pdf
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spelling	my-upm-ir.604432018-04-26T03:42:44Z Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2 2015-02 Kalyanasundram, Jeevanathan Food and commensal lactic acid bacteria (LAB) surface display system exploitation for bacterial, viral, or protozoal antigen delivery has received immense interest currently. The Generally Regarded as Safe (GRAS) status of LAB such as Lactococcus lactis coupled with non-recombinant strategy of in-trans surface display system, provide a safe platform for therapeutic drug and vaccine development. However, therapeutic proteins fused with cell-wall anchoring motif production are predominantly limited to prokaryotic expression system. This presents a major disadvantage in surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study,glycosylated murine Tyrosinase related protein-2, mTRP-2, tumor associated antigen anchoring to L. lactis cell wall was attempted. The mtrp-2-cA (AcmA, peptidoglycan anchoring motif) fusion gene expression in Chinese Hamster Ovary, CHO cells was carried out. Initial CHO cell expression of both native mtrp-2 and mtrp-cA was a failure. Codon optimized mtrp-2 and cA genes also did not result in target protein production. In order to investigate post-translational modification interruption, expression of codon optimized mtrp-2125-276 epitope devoid of mtrp-2 native maturation signal peptide was performed which resulted in misfolded plus aggregated mTRP-2125-276 and mTRP-2125-276 –cA protein production. Successful expression of both mtrp-2125-276 and mtrp-2125-276 -cA genes suggest CHO cell’s endoplasmic reticulum signal peptidase inability to recognize mTRP-2 signal peptide cleavage site. The following substitution of native mTRP-2 signal peptide with Chinese Hamster TRP-2 signal peptide, CHsp resolved this issue by successful expression of soluble mTRP-2 and mTRP-2-cA by CHO cells in both intracellular and extracellular fraction. A total amount of 40 μg of mTRP-2-cA protein from 2.7 g in wet weight of CHO cells was purified and detected to be glycosylated by glycoprotein staining. Subsequent mTRP-2-cA anchoring to the cell wall of L. lactis showed excitation of FITC conjugate on secondary antibody which signified successful binding of glycosylated TRP-2 on the surface of L. lactis. Glycosylation Therapeutics - Research 2015-02 Thesis http://psasir.upm.edu.my/id/eprint/60443/ http://psasir.upm.edu.my/id/eprint/60443/1/FBSB%202015%2013IR.pdf text en public masters Universiti Putra Malaysia Glycosylation Therapeutics - Research
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Glycosylation Therapeutics - Research
spellingShingle	Glycosylation Therapeutics - Research Kalyanasundram, Jeevanathan Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
description	Food and commensal lactic acid bacteria (LAB) surface display system exploitation for bacterial, viral, or protozoal antigen delivery has received immense interest currently. The Generally Regarded as Safe (GRAS) status of LAB such as Lactococcus lactis coupled with non-recombinant strategy of in-trans surface display system, provide a safe platform for therapeutic drug and vaccine development. However, therapeutic proteins fused with cell-wall anchoring motif production are predominantly limited to prokaryotic expression system. This presents a major disadvantage in surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study,glycosylated murine Tyrosinase related protein-2, mTRP-2, tumor associated antigen anchoring to L. lactis cell wall was attempted. The mtrp-2-cA (AcmA, peptidoglycan anchoring motif) fusion gene expression in Chinese Hamster Ovary, CHO cells was carried out. Initial CHO cell expression of both native mtrp-2 and mtrp-cA was a failure. Codon optimized mtrp-2 and cA genes also did not result in target protein production. In order to investigate post-translational modification interruption, expression of codon optimized mtrp-2125-276 epitope devoid of mtrp-2 native maturation signal peptide was performed which resulted in misfolded plus aggregated mTRP-2125-276 and mTRP-2125-276 –cA protein production. Successful expression of both mtrp-2125-276 and mtrp-2125-276 -cA genes suggest CHO cell’s endoplasmic reticulum signal peptidase inability to recognize mTRP-2 signal peptide cleavage site. The following substitution of native mTRP-2 signal peptide with Chinese Hamster TRP-2 signal peptide, CHsp resolved this issue by successful expression of soluble mTRP-2 and mTRP-2-cA by CHO cells in both intracellular and extracellular fraction. A total amount of 40 μg of mTRP-2-cA protein from 2.7 g in wet weight of CHO cells was purified and detected to be glycosylated by glycoprotein staining. Subsequent mTRP-2-cA anchoring to the cell wall of L. lactis showed excitation of FITC conjugate on secondary antibody which signified successful binding of glycosylated TRP-2 on the surface of L. lactis.
format	Thesis
qualification_level	Master's degree
author	Kalyanasundram, Jeevanathan
author_facet	Kalyanasundram, Jeevanathan
author_sort	Kalyanasundram, Jeevanathan
title	Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
title_short	Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
title_full	Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
title_fullStr	Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
title_full_unstemmed	Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
title_sort	construction and characterization of a lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2
granting_institution	Universiti Putra Malaysia
publishDate	2015
url	http://psasir.upm.edu.my/id/eprint/60443/1/FBSB%202015%2013IR.pdf
_version_	1747812272876552192

Construction and characterization of a Lactococcus lactis in-trans surface display system harboring murine glycosylated tyrosinase related protein-2

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