Recombinant destabilase-lysozyme: synthesis de novo in E. coli and action mechanism of the enzyme expressed in Spodoptera frugiperda.
Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.
Zavalova
L L
LL
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 119997, Russia. leech@humgen.siobc.ras.ru
Baskova
I P
IP
Barsova
E V
EV
Snezhkov
E V
EV
Akopov
S B
SB
Lopatin
S A
SA
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D047709:Hirudo medicinalis / Q000201:enzymology; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D009113:Muramidase; D010450:Endopeptidases; C045348:fibrin destabilase; D000117:Acetylglucosamine