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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Turner P,Holst O,Karlsson ENdoi
10.1016/j.pep.2004.09.012keywords:
subject
Has Abstractpub_date
2005-01-01 00:00:00pages
54-60issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(04)00327-4journal_volume
39pub_type
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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