Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12
Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification...
Published in: | Enzyme Research |
---|---|
Main Authors: | , , , , |
Format: | Text |
Language: | English |
Published: |
Hindawi Publishing Corporation
2014
|
Subjects: | |
Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100274 http://www.ncbi.nlm.nih.gov/pubmed/25093119 https://doi.org/10.1155/2014/197938 |
id |
ftpubmed:oai:pubmedcentral.nih.gov:4100274 |
---|---|
record_format |
openpolar |
spelling |
ftpubmed:oai:pubmedcentral.nih.gov:4100274 2023-05-15T13:58:32+02:00 Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 Alias, Norsyuhada Ahmad Mazian, Mu'adz Salleh, Abu Bakar Basri, Mahiran Rahman, Raja Noor Zaliha Raja Abd. 2014 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100274 http://www.ncbi.nlm.nih.gov/pubmed/25093119 https://doi.org/10.1155/2014/197938 en eng Hindawi Publishing Corporation http://www.ncbi.nlm.nih.gov/pmc/articles/PMC http://www.ncbi.nlm.nih.gov/pubmed/25093119 http://dx.doi.org/10.1155/2014/197938 Copyright © 2014 Norsyuhada Alias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CC-BY Research Article Text 2014 ftpubmed https://doi.org/10.1155/2014/197938 2014-08-10T00:45:17Z Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. Text Antarc* Antarctic Antarctica PubMed Central (PMC) Antarctic Enzyme Research 2014 1 20 |
institution |
Open Polar |
collection |
PubMed Central (PMC) |
op_collection_id |
ftpubmed |
language |
English |
topic |
Research Article |
spellingShingle |
Research Article Alias, Norsyuhada Ahmad Mazian, Mu'adz Salleh, Abu Bakar Basri, Mahiran Rahman, Raja Noor Zaliha Raja Abd. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
topic_facet |
Research Article |
description |
Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. |
format |
Text |
author |
Alias, Norsyuhada Ahmad Mazian, Mu'adz Salleh, Abu Bakar Basri, Mahiran Rahman, Raja Noor Zaliha Raja Abd. |
author_facet |
Alias, Norsyuhada Ahmad Mazian, Mu'adz Salleh, Abu Bakar Basri, Mahiran Rahman, Raja Noor Zaliha Raja Abd. |
author_sort |
Alias, Norsyuhada |
title |
Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
title_short |
Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
title_full |
Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
title_fullStr |
Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
title_full_unstemmed |
Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12 |
title_sort |
molecular cloning and optimization for high level expression of cold-adapted serine protease from antarctic yeast glaciozyma antarctica pi12 |
publisher |
Hindawi Publishing Corporation |
publishDate |
2014 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100274 http://www.ncbi.nlm.nih.gov/pubmed/25093119 https://doi.org/10.1155/2014/197938 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic Antarctica |
genre_facet |
Antarc* Antarctic Antarctica |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC http://www.ncbi.nlm.nih.gov/pubmed/25093119 http://dx.doi.org/10.1155/2014/197938 |
op_rights |
Copyright © 2014 Norsyuhada Alias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.1155/2014/197938 |
container_title |
Enzyme Research |
container_volume |
2014 |
container_start_page |
1 |
op_container_end_page |
20 |
_version_ |
1766266875987099648 |