Heterologous hyper-expression of a glucansucrase-type glycosyltransferase gene

Heterologous expression of the large glucansucrase-type glycosyltransferases genes is still a challenge, and typically yields are poor. Therefore, a number of different Escherichia coli systems for the expression of such a gene, encoding the glycosyltransferase R (GtfR) from Streptococcus oralis, we...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2008-05, Vol.79 (2), p.255-261
Main Authors: Swistowska, Anna Maria, Wittrock, Sabine, Collisi, Wera, Hofer, Bernd
Format: Article
Language:English
Subjects:
Binding sites
Biological and medical sciences
Biosynthesis
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Cloning
Construction
E coli
Enzyme kinetics
Enzymes
Escherichia coli
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gene Expression
Genetic engineering
Genetic technics
Glucansucrase
Glucosyltransferase
Glycosyltransferases - chemistry
Glycosyltransferases - genetics
Glycosyltransferases - metabolism
High level expression
Life Sciences
Methods. Procedures. Technologies
Microbial Genetics and Genomics
Microbiology
Modification of gene expression level
Plasmids
Streptococcus oralis
Streptococcus oralis - enzymology
Streptococcus oralis - genetics
Streptococcus oralis - metabolism
Studies
Truncation
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Summary:Heterologous expression of the large glucansucrase-type glycosyltransferases genes is still a challenge, and typically yields are poor. Therefore, a number of different Escherichia coli systems for the expression of such a gene, encoding the glycosyltransferase R (GtfR) from Streptococcus oralis, were constructed and evaluated. We thereby obtained a strain producing the highest molar yields described so far for this class of enzymes. Cloning of a 5'-terminally truncated version of the gene in the expression vector pET33b(+) yielded, in dissolved form, about 2 μmol (300 mg) of enzyme per liter of culture of an optical density at 600 nm of four. Problems frequently encountered in the heterologous biosynthesis of this class of enzymes, such as formation of a high fraction of insoluble aggregates and/or proteolytic degradation, were not observed in the described system. The over-produced enzyme, devoid of almost its entire variable region, retained its characteristic activities.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-008-1435-0