Relationship between the optimal temperature for oxidative refolding and the thermal stability of refolded state of hen lysozyme three-disulfide derivatives.
The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Tachibana
H
H
Department of Biology, Faculty of Science, Kobe University, Japan.
Ohta
K
K
Sawano
H
H
Koumoto
Y
Y
Segawa
S
S
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D004220:Disulfides; D011994:Recombinant Proteins; D009113:Muramidase; D005990:Glycerol