Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.

ecombinant protein expression is very important in biotechnology. Successful protein expression depends on the expression host, vector and target protein. Escherichia coli being a popular expression host, is still plagued by various problems in expression like formation of inclusion bodies, incorrec...

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Main Author:	Velaithan, Vithya
Format:	Thesis
Language:	English English
Published:	2011
Subjects:	Escherichia coli Recombinant proteins Peptides
Online Access:	http://psasir.upm.edu.my/id/eprint/27854/1/FBSB%202011%2049R.pdf
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spelling	my-upm-ir.278542014-05-05T01:27:34Z Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli. 2011-09 Velaithan, Vithya ecombinant protein expression is very important in biotechnology. Successful protein expression depends on the expression host, vector and target protein. Escherichia coli being a popular expression host, is still plagued by various problems in expression like formation of inclusion bodies, incorrect folding and low soluble protein yield. These problems can be circumvented by using different promoters, different host strains, co-expression of chaperones, reduction of culture temperature and secretion of proteins into periplasm and culture medium. In this study, periplasmic protein secretion was investigated by using novel synthetic signal peptides, spA and spD. The two signal peptides were designed based on signal peptides of Bacillus spp. for secretion of heterologous proteins. They were amplified and ligated to genes coding for the green fluorescent protein (GFP) and cyclodextrin lucanotransferase (CGTase) to construct secretion cassettes spAGFP, spDGFP and spACGT and spDCGT. These secretion cassettes were first cloned into pCR®-Blunt II-TOPO cloning vector. The cassettes were then sub-cloned into pET-32b(+) expression vector to construct pAGFP, pDGFP, pACGT and transformed into competent E. coli BL21(DE3) and BL21(DE3)pLysS cells. The cloning of secretion cassette spD*CGT into pET-32b(+) was not successful. GFP without the signal peptide was cloned into similar pET-32b(+) as a control and the construct was named pGFP. SDS-PAGE and western blotting results for recombinant GFP clones showed successful expression and secretion into the periplasm. Fluorescence analysis for GFP clones showed that the secreted GFP was not fluorescent while cytoplasmically expressed GFP was fluorescent. Induction temperature also affected the secretion of recombinant GFP as better secretion was attained at 37°C. SDS-PAGE analysis for recombinant clone BLpACGT showed that CGTase was detected in both cytoplasm and periplasm but in Western blotting only cytoplasmic expression was detected. However, the positive control, (CGTase with native signal peptide) was detected in both cytoplasm and periplasm. Cell growth analysis for recombinant clones GFP and CGTase did not show any adverse effect to the secretion host. These results show that the synthetic signal peptides, spA and spD, could direct recombinant proteins to the periplasmic space Escherichia coli Recombinant proteins Peptides 2011-09 Thesis http://psasir.upm.edu.my/id/eprint/27854/ http://psasir.upm.edu.my/id/eprint/27854/1/FBSB%202011%2049R.pdf application/pdf en public masters Universiti Putra Malaysia Escherichia coli Recombinant proteins Peptides Faculty of Biotechnology and Biomolecular Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Escherichia coli Recombinant proteins Peptides
spellingShingle	Escherichia coli Recombinant proteins Peptides Velaithan, Vithya Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
description	ecombinant protein expression is very important in biotechnology. Successful protein expression depends on the expression host, vector and target protein. Escherichia coli being a popular expression host, is still plagued by various problems in expression like formation of inclusion bodies, incorrect folding and low soluble protein yield. These problems can be circumvented by using different promoters, different host strains, co-expression of chaperones, reduction of culture temperature and secretion of proteins into periplasm and culture medium. In this study, periplasmic protein secretion was investigated by using novel synthetic signal peptides, spA and spD. The two signal peptides were designed based on signal peptides of Bacillus spp. for secretion of heterologous proteins. They were amplified and ligated to genes coding for the green fluorescent protein (GFP) and cyclodextrin lucanotransferase (CGTase) to construct secretion cassettes spAGFP, spDGFP and spACGT and spDCGT. These secretion cassettes were first cloned into pCR®-Blunt II-TOPO cloning vector. The cassettes were then sub-cloned into pET-32b(+) expression vector to construct pAGFP, pDGFP, pACGT and transformed into competent E. coli BL21(DE3) and BL21(DE3)pLysS cells. The cloning of secretion cassette spD*CGT into pET-32b(+) was not successful. GFP without the signal peptide was cloned into similar pET-32b(+) as a control and the construct was named pGFP. SDS-PAGE and western blotting results for recombinant GFP clones showed successful expression and secretion into the periplasm. Fluorescence analysis for GFP clones showed that the secreted GFP was not fluorescent while cytoplasmically expressed GFP was fluorescent. Induction temperature also affected the secretion of recombinant GFP as better secretion was attained at 37°C. SDS-PAGE analysis for recombinant clone BLpACGT showed that CGTase was detected in both cytoplasm and periplasm but in Western blotting only cytoplasmic expression was detected. However, the positive control, (CGTase with native signal peptide) was detected in both cytoplasm and periplasm. Cell growth analysis for recombinant clones GFP and CGTase did not show any adverse effect to the secretion host. These results show that the synthetic signal peptides, spA and spD, could direct recombinant proteins to the periplasmic space
format	Thesis
qualification_level	Master's degree
author	Velaithan, Vithya
author_facet	Velaithan, Vithya
author_sort	Velaithan, Vithya
title	Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
title_short	Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
title_full	Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
title_fullStr	Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
title_full_unstemmed	Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.
title_sort	development of secretory expression system using synthetic signal peptides spa and spd for protein production in escherichia coli.
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2011
url	http://psasir.upm.edu.my/id/eprint/27854/1/FBSB%202011%2049R.pdf
_version_	1747811602216779776

Development of secretory expression system using synthetic signal peptides spA and spD for protein production in Escherichia coli.

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