Fusion tags and chaperone co-expression modulate both the solubility and the inclusion body features of the recombinant CLIPB14 serine protease.
Chaperone co-expression and the fusion to different tags were used to modify the aggregation pattern of the putative serine protease CLIPB14 precipitated in Escherichia coli inclusion bodies. A set of common tags used in expression vectors has been selected, as well as two bacterial strains over-expressing the chaperones GroELS and ibpA/B, respectively. The presence of the fused tags resulted in an improved solubility of CLIPB14 but also in a higher presence of contaminants in the inclusion bodies, while chaperone co-expression promoted the binding of all the chaperone machinery involved into the disaggregation to the CLIPB14. Furthermore, each tag influenced in a specific manner the re-aggregation of the denatured CLIPB14 constructs during urea dilution and the preliminary trials indicated that the CLIPB14 fusions with higher homogeneity and lower re-aggregation rate were the optimal candidates for refolding assays. In conclusion, it is possible to tune the quality of the inclusion bodies by choosing the suitable combination of tag and chaperone co-expression that minimize the non-productive side reactions during refolding.
Schrödel
Andrea
A
EMBL, Meyerhofstr. 1, D-69117 Heidelberg, Germany.
Volz
Jennifer
J
de Marco
Ario
A
D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D020224:Expressed Sequence Tags / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D002479:Inclusion Bodies / Q000201:enzymology* / Q000648:ultrastructure*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases