Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.
Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.
Scientific Research Institute of Physical-Chemical Medicine of the Federal Medical and Biological Agency, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia; Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudny, Moscovskaya obl 141700, Russia. Electronic address: vmanuvera@yandex.ru.
Valentin A
VA
Manuvera
Scientific Research Institute of Physical-Chemical Medicine of the Federal Medical and Biological Agency, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
Alexey S
AS
Kurdyumov
Scientific Research Institute of Physical-Chemical Medicine of the Federal Medical and Biological Agency, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
Kseniya A
KA
Filonova
Scientific Research Institute of Physical-Chemical Medicine of the Federal Medical and Biological Agency, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia; Moscow Institute of Physics and Technology, Institutskiy per. 9, Dolgoprudny, Moscovskaya obl 141700, Russia.
Vassili N
VN
Lazarev
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001424:Bacterial Infections / Q000188:drug therapy; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D047709:Hirudo medicinalis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000900:Anti-Bacterial Agents; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D009113:Muramidase; D010450:Endopeptidases; C045348:fibrin destabilase
Destabilase-lysozyme; E. coli; Expi293F; Isopeptidase; Medicinal leeches; P. pastoris