Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris
We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP...
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Association of Chemical Engineers of Serbia
2015-01-01
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doaj-bf4269305a5241b5bfc961ad0c6afab72020-11-25T00:10:00ZengAssociation of Chemical Engineers of SerbiaHemijska Industrija 0367-598X2217-74262015-01-0169668970110.2298/HEMIND140823003T0367-598X1500003TCloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastorisTadić Vojin M.0Balaž Ana Marija J.1Petrić Marija P.2Milošević Snežana M.3Zelenović Nevena D.4Raspor Martin Z.5Tadić Jovan M.6Prodanović Radivoje M.7Institute for Biological Research “Siniša Stanković”, Department for Plant Physiology, BelgradeFaculty of Chemistry, Department for Biochemistry, BelgradeInstitute for Biological Research “Siniša Stanković”, Department for Plant Physiology, BelgradeInstitute for Biological Research “Siniša Stanković”, Department for Plant Physiology, BelgradeInstitute for Chemistry, Technology and Metallurgy, BelgradeInstitute for Biological Research “Siniša Stanković”, Department for Plant Physiology, BelgradeCarnegie Institution for Science, Department of Global Ecology, Stanford, USAFaculty of Chemistry, Department for Biochemistry, BelgradeWe have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5α strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme further studies on purification and refolding of expressed protein are necessary.http://www.doiserbia.nb.rs/img/doi/0367-598X/2015/0367-598X1500003T.pdfSaccharomyces cerevisiaePichia pastoriscarbohydrate oxidaseglycosylation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Tadić Vojin M. Balaž Ana Marija J. Petrić Marija P. Milošević Snežana M. Zelenović Nevena D. Raspor Martin Z. Tadić Jovan M. Prodanović Radivoje M. |
spellingShingle |
Tadić Vojin M. Balaž Ana Marija J. Petrić Marija P. Milošević Snežana M. Zelenović Nevena D. Raspor Martin Z. Tadić Jovan M. Prodanović Radivoje M. Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris Hemijska Industrija Saccharomyces cerevisiae Pichia pastoris carbohydrate oxidase glycosylation |
author_facet |
Tadić Vojin M. Balaž Ana Marija J. Petrić Marija P. Milošević Snežana M. Zelenović Nevena D. Raspor Martin Z. Tadić Jovan M. Prodanović Radivoje M. |
author_sort |
Tadić Vojin M. |
title |
Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris |
title_short |
Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris |
title_full |
Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris |
title_fullStr |
Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris |
title_full_unstemmed |
Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris |
title_sort |
cloning of the gene for a carbohydrate oxidase from lactuca sativa in the yeasts saccharomyces cerevisiae and pichia pastoris |
publisher |
Association of Chemical Engineers of Serbia |
series |
Hemijska Industrija |
issn |
0367-598X 2217-7426 |
publishDate |
2015-01-01 |
description |
We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in
two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The
synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned
into the vector pUC57 and inserted into plasmids pYES2 and pGAP using
Escherichia coli DH5α strain. The P. pastoris strain X-33 and the S.
cerevisiae strain InvSC1 were used for extracellular expression of CHO. After
transformation of P. pastoris X-33 with CHO-pGAP construct none of the
colonies showed CHO activity. Two samples displayed a band which did not
exist in the sample with the empty vector similar to the molecular weight of
CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES
constructs. Three colonies grew on the plate with cells transformed with the
construct. One of the samples showed a band corresponding to about 110 kDa,
but no CHO activity was recorded in this case either. Cloning of the foreign
genes and heterologous expression in yeasts is widely used in biotechnology,
but sometimes can be very dependent on the gene sequence and strain used. In
order to obtain active CHO enzyme further studies on purification and
refolding of expressed protein are necessary. |
topic |
Saccharomyces cerevisiae Pichia pastoris carbohydrate oxidase glycosylation |
url |
http://www.doiserbia.nb.rs/img/doi/0367-598X/2015/0367-598X1500003T.pdf |
work_keys_str_mv |
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