Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits.
Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies. The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts. The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series. Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined. Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties. Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea.
Lim
W K
WK
Biology Department, University of Alabama, Tuscaloosa 35487-0344.
Smith-Somerville
H E
HE
Hardman
J K
JK
D001965:Bromides / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D005786:Gene Expression Regulation; D002479:Inclusion Bodies / Q000648:ultrastructure; D008094:Lithium / Q000494:pharmacology; D018020:Lithium Compounds; D009154:Mutation; D009566:Nitrates / Q000494:pharmacology; D012974:Sodium Iodide / Q000494:pharmacology; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000032:analysis; D013861:Thiocyanates / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D001965:Bromides; D018020:Lithium Compounds; D009566:Nitrates; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; C040949:lithium bromide; C031618:sodium nitrate; D008094:Lithium; D014367:Tryptophan Synthase; D012974:Sodium Iodide; D002122:Calcium Chloride; C009941:potassium thiocyanate