Removal of DnaK contamination during fusion protein purifications
Section snippets
DnaK binding site prediction
Identification of possible DnaK binding sites on the linker regions of the different vectors was performed as described previously [2], using the algorithm of Rüdiger et al. [3]. These authors have identified DnaK binding sites by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins [3]. The algorithm attributes overall statistical energy contributions (ΔΔGK) to peptides of 13 adjacent amino acids. The lower the value obtained for a specific
Results and discussion
We have analyzed the connector peptide regions of several pET and pGEX vectors using a predicting algorithm for DnaK binding motifs within protein sequences [3]. This algorithm analyzes 13-mer peptides consisting in a core of five residues and two flanking regions of four residues each. The algorithm attributes overall statistical energy contributions (ΔΔGK) to the 13 adjacent amino acid windows. The lower the value obtained for a specific segment, the higher the predicted affinity for DnaK.
Acknowledgements
This work was supported by grants from CONICET, UNR, and Foncyt, Argentina. EAC is member of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). D.V.R. is a Fellow of the same institution.
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