2.1.4. Octyl-POE Extraction

To obtain the fraction with the highest content of heat-modifiable OM proteins, a stepwise extraction of the cell walls of *M. primoryensis* KMM 3633T with a POE solution was carried out, as is known, selectively extracting OM proteins of gram-negative bacteria [25]. It was found that polypeptides changing their molecular weight when heated under denaturing conditions are extracted at different concentrations of POE in the range from 0.5 to 3.0% by weight [25]. In the case of *M. primoryensis* KMM 3633T, the greatest amount of the heat-modifiable proteins was obtained using 0.5% detergent solution, so the extraction was repeated three times. According to the SDS-PAGE data an electrophoretically homogeneous protein was isolated in the oligomeric form as a result of purification of the obtained sum fraction by gel permeation chromatography on Sephacryl S-300 (Figure 1B, lane 4). After boiling in SDS solution the protein band observed migrated to the same position as MpOmp monomer isolated with Sarcosyl extraction (Figure 1B, lane 3).

It should be noted that in this case, the unheated protein migrated to a position corresponding to MW of about 72 kDa. This apparent MW value was lower than that of the MpOmp oligomer isolated by Sarcosyl extraction. The cause of this phenomenon could be the presence of residual amount of POE in the protein sample. A similar shift of the protein MW was observed for VhOmp of *Vibrio harveyi* in [26].

Thus, according to the SDS-PAGE data, MW the protein isolated with Sarcosyl extraction was identical to that of the protein obtained by the Garavito method [25]. Taking into account the data obtained, in subsequent experiments a sample obtained as a result of the removal of cytoplasmic proteins with Sarcosyl was used. We chose this method of the target protein isolation because of the simplicity and at the same time the high selectivity of the isolation procedure.

**Figure 1.** Purification and temperature denaturation of *M. primoryensis* KMM 3633T porin (MpOmp): (**A**): Crude cell envelope fraction (crude membrane) of *M. primoryensis* KMM 3633T (2, 3); marker proteins (1). Sample 3 is heated at 99 ◦C. (**B**): MpOmp isolated from the crude membrane fraction of *M. primoryensis KMM* 3633<sup>T</sup> after treatment with 1% Sarcosyl (1, 2); MpOmp isolated from combined OM protein fraction obtained as a result of three time extraction with 0.5% POE of *M. primoryensis* KMM 3633<sup>T</sup> cell envelope and gel permeation chromatography on Sephacryl S-300 (3, 4); marker proteins (5). Samples 2 and 3 are heated at 99 ◦C. (**C**): MpOmp isolated with Sarcosyl extraction (1–5); samples 2, 3, 4, and 5 were heated at 30, 40, 50, and 99 ◦C, respectively; MpOmp isolated with POE extraction (6–10); samples 7, 8, 9, and 10 were heated at 30, 40, 50, and 99 ◦C, respectively; marker proteins (11).
