In vitro refolding of cyclomaltodextrin glucanotransferase from cytoplasmic inclusion bodies formed upon expression in Escherichia coli.
The recovery of active cyclomaltodextrin glucanotransferase (CGT) from inclusion bodies formed upon expression of a signal sequence deletion mutant (cgt delta ss) gene in Escherichia coli was studied. Under the conditions examined the in vitro renaturation yield of active enzyme was up to 81% of the maximum activity from urea solubilized and up to 3.7% from guanidinium hydrochloride (GdmCl) solubilized inclusion bodies. Refolding from GdmCl solutions resulted in the reaggregation of delta ss CGT. Although native (folded) CGT showed enzymatic activity at urea concentrations as high as 10 M, the inclusion bodies could be solubilized with 3 M urea solution, but the preparation had no enzymatic activity prior to reduction of the denaturant concentration. This suggests that the delta ssCGT inclusion bodies are composed of molecules that are trapped in an inactive state. The individual molecules may have extensive secondary structure, since inclusion bodies solubilized with 4.5 M urea gave maximum yield of activity in the renaturation step. With higher urea concentration the yield decreased. Thus, a "mild" solubilization technique seems to increase the yield of active delta ssCGT from the inclusion bodies. The stability of the refolded delta ssCGT was identical to that of extracellularly produced wild type CGT, whereas a disulfide bond mutant, Ser-70-CGT, showed reduced stability under identical conditions. This suggests that the single disulfide bond in delta ssCGT is formed during refolding and contributes to the stability of the molecule. A fusion of maltose binding protein to the NH2-terminus of delta ssCGT did not significantly affect the in vitro folding of the delta ssCGT portion when tested by a denaturation-renaturation experiment.
Hellman
J
J
Department of Biochemistry, University of Turku, Finland.
Lassila
P
P
Mäntsälä
P
P
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005964:Glucosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase