*3.1. Isolation Procedure of OM Protein from M. primoryensis KMM 3633<sup>T</sup>*

To date several advances have been made in the development of isolation methods of OM pore-forming proteins. Since porins as integral proteins exist in an intrinsically anisotropic environment within the bacterial membrane the main condition for maximizing protein extraction is the selection of a suitable detergent [25]. Unlike terrestrial, marine bacteria cell envelope is not tightly associated with rigid peptidoglycan layer and therefore the OM may be easily separated [37,38]. Thus, the standard isolation and purification procedure used for OM porins of gram-negative bacteria is not exactly applicable for marine OM proteins. In the case of marine bacteria, the OM proteins may be isolated with the sequential extraction of the microbial cells with solutions of high ionic strength, even without previous cell destruction [39]. In light of the above special attention has been given by us to the choice of cell envelope isolation condition and the selection of a suitable detergent for the extraction of *M. primoryensis* KMM 3633<sup>T</sup> OM proteins.

To determine the influence of the cultivation temperature on the protein composition of the cell envelope of the psychrophilic bacterium *M. primoryensis* KMM 3633T, the bacterial cells were grown at a temperature of 0, 6–8, and 24 ◦C using the growth medium given in [23]. When growing microorganisms at low temperatures, the most intense polypeptide band was observed in the region corresponding to the molecular masses of the porins of terrestrial bacteria. Taking these results into account, for the further study we selected the *M. primoryensis* KMM 3633T cells, cultivated at 6–8 ◦C under aerobic conditions.

In the electrophoretic profile of the crude membrane fraction obtained by ultrasonic disintegration, proteins that changed their electrophoretic mobility after heat pre-treatment of the protein sample were detected. This allows us to assume that heat-modifiable proteins are present in the cell lysate of *M. primoryensis* KMM 3633T.

To isolate the OM protein fraction from the cell envelope of *M. primoryensis* KMM 3633T, two methods were tested, commonly used to obtain OM proteins from the terrestrial bacteria. One of them consists in the step-by-step extraction of cell membranes obtained after ultrasound treatment with a nonionic detergent POE [25]. By the author data subsequent extraction with POE allows to yield several fractions highly enriched in *E. coli* porins.

The second method is based on the removal of cytoplasmic proteins from the cell envelope fraction with nonionic detergent Sarcosyl extraction. As known, Sarcosyl is commonly used in the purification procedure of OM proteins of gram-negative terrestrial bacteria, and this method produces samples free of cytoplasmic proteins. OM proteins in this case remain in the sediment. As we have seen earlier, after Sarcosyl pre-treatment the fraction of OM proteins of *Yersinia ruckeri* practically did not contain low molecular weight cytoplasmic proteins [40]. However, in the case of OM proteins of *M. primoryensis KMM* 3633T in the Sarcosyl-soluble fraction, in addition to cytoplasmic proteins, we found the presence of a certain amount of OM proteins with a molecular weight of 30–50 kDa. This result indicates that the OM proteins of *M. primoryensis* KMM 3633T are partially soluble in Sarcosyl.
