Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration.
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.
Turner
Pernilla
P
Department of Biotechnology, Lund University, SE-221 00 Lund, Sweden. pernilla.turner@biotek.lu.se
Holst
Olle
O
Karlsson
Eva Nordberg
EN
D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase