Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange.
In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.
Imoto
T
T
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Yamada
H
H
Yasukochi
T
T
Yamada
E
E
Ito
Y
Y
Ueda
T
T
Nagatani
H
H
Miki
T
T
Horiuchi
T
T
D000409:Alanine / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D010084:Oxidation-Reduction; D010957:Plasmids; D013438:Sulfhydryl Compounds; D014633:Valine / Q000235:genetics*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D004247:DNA; D009113:Muramidase; D014633:Valine; D000409:Alanine