Advanced

Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration

Turner, Pernilla LU ; Holst, Olle LU and Nordberg Karlsson, Eva LU (2005) In Protein Expression and Purification 39(1). p.54-60
Abstract
Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be optimized. We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus. One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA. The enzyme... (More)
Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be optimized. We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus. One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA. The enzyme activity could be increased 6-7 times solely by finely tuning the IPTG concentration, but the activity level was very sensitive to the amount of inducer applied. Hence, the IPTG concentration may have to be optimized for every protein under the conditions used. The fusion protein-strategy gave a slightly lower total activity but the level of soluble recombinant protein obtained was in this case significantly less sensitive to the inducer concentration applied. Moreover, the activity could be increased about 2-fold by cleaving off the solubility-tag (NusA) by enterokinase. (C) 2004 Elsevier Inc. All rights reserved. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
39
issue
1
pages
54 - 60
publisher
Academic Press
external identifiers
  • pmid:15596360
  • wos:000226153600007
  • scopus:10644275363
ISSN
1046-5928
DOI
10.1016/j.pep.2004.09.012
language
English
LU publication?
yes
id
352b76a9-8970-41a7-8bea-76d5feb8820a (old id 155217)
date added to LUP
2007-07-03 09:10:03
date last changed
2017-08-27 04:11:33
@article{352b76a9-8970-41a7-8bea-76d5feb8820a,
  abstract     = {Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be optimized. We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus. One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA. The enzyme activity could be increased 6-7 times solely by finely tuning the IPTG concentration, but the activity level was very sensitive to the amount of inducer applied. Hence, the IPTG concentration may have to be optimized for every protein under the conditions used. The fusion protein-strategy gave a slightly lower total activity but the level of soluble recombinant protein obtained was in this case significantly less sensitive to the inducer concentration applied. Moreover, the activity could be increased about 2-fold by cleaving off the solubility-tag (NusA) by enterokinase. (C) 2004 Elsevier Inc. All rights reserved.},
  author       = {Turner, Pernilla and Holst, Olle and Nordberg Karlsson, Eva},
  issn         = {1046-5928},
  language     = {eng},
  number       = {1},
  pages        = {54--60},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration},
  url          = {http://dx.doi.org/10.1016/j.pep.2004.09.012},
  volume       = {39},
  year         = {2005},
}