Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange

Taiji Imoto, Hidenori Yamada, Takanori Yasukochi, Eiichi Yamada, Yuji Ito, Tadashi Ueda, Hiroko Nagatani, Takeyoshi Miki, Tadao Horiuchi

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28 Citations (Scopus)

Abstract

In the preceding paper in this issue, we described the over production of one mutant chicken lysozyme in Escherichia coil. Since this lysozyme contained two amino acid substitutions (Ala31→Valand 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-1 Val31Ser106-, Met-1 Ser106-,Met-1 Val31-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°C, follow ed by the specific activity measurements of the folded en zymes, 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.

Original languageEnglish
Pages (from-to)333-338
Number of pages6
JournalProtein Engineering, Design and Selection
Volume1
Issue number4
DOIs
Publication statusPublished - Aug 1987

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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