Optimized Fractioning and Structure Analysis of the Reactivating Factor from Luteococcus japonicus subsp. casei

Abstract

The chemical structure of the extracellular reactivating factor (RF) from Luteococcus japonicus subsp. casei was determined; this factor promotes survival of a small subpopulation of the producer cells under lethal stress impact. For the isolation and purification of this RF, the previously developed method for RF isolation from Saccharomyces cerevisiae was optimized. A total of 15 fractions were obtained from the culture liquid of Luteococcus casei, two of which (I and IV) exhibited reactivation activity against the cells subjected to a lethal stress impact (UV irradiation). The method included solid-phase extraction of the peptides on a hydrophobic sorbent with the C₈ phase and subsequent multistage separation using RP-HPLC. Mass spectral analysis (MALDI-TOF) was used to determine the molecular characteristics of fraction IV. Efficient ionization was not achieved for fraction I. Mass charges for fraction IV were 773.394 and 788.102 Da. Edman automatic sequencing was used to identify these components as peptides: Ala-Pro-Asn-Glu-Asn-Gln-Gly and Ala-Pro-Asn-Glu-Glu-Gln-Gly. No similarity to any known full-size functional peptide molecules in the databases on polypeptide primary structures was revealed. Formation of biologically active peptides by L. casei may be associated with non-template synthesis and probably involves proteolysis of a large protein.