1970;227:680C685

1970;227:680C685. subsequent removal of the tags necessary for chromatography of these proteins by specific proteases. One of the widely used options is the introduction of thrombin recognition sites. Thrombin (coagulation factor II) is usually a component Lerociclib dihydrochloride of the mammalian blood coagulation system and functions as a site specific serine protease, insensitive to Lerociclib dihydrochloride buffer solutions made up of various detergents [1]. This protein recognizes the LVPR^GS site and introduces a gap between arginine and glycine [2]. To study the genesis of HlyII in bacterial cells and the conversation of the toxin with eukaryotic cells, an MA panel was created for the C-terminal domain name of the pore-forming protein HlyII [3]. This work is usually devoted to the descrition of CDKN2A HlyIIC-15, which binds efficiently to the thrombin site of the fusion protein, but not to other peptide sites of the recombinant CTD. RESULTS Characterization of the amino acid sequence HlyIICTD. One of the virulence factors of an opportunistic microorganism is the pore-forming toxin hemolysin II (HlyII) [4]. Hemolysin II with a -barrel type structure has high homology with alpha-toxin and differs from it by the presence of a C-terminal amino acid excess of 94 residues [5]. This region in NMR analysis demonstrated a unique spatial structure not previously described [6]. The gene region was cloned and HlyIICTD was purified as described in the work of Rudenko et al. [3]. The amino acid sequence of HlyIICTD consists of the amino acid residues of the C-terminal redundancy of HlyII, a thrombin site, six histidine residues, and a linker (Fig. 1). Open in a separate windows Fig. 1. Amino acid sequence of HlyIICTD cloned from HlyII ATCC 14579T in pET29b. The molecular weight of the cloned CTD is usually 13?906.59 Da. The thrombin site is usually highlighted in strong letters, / indicates the site of proteolytic attack by thrombin. Italics indicate the area with six histidines. Features of the conversation of HlyIICTD with erythrocytes, detected by MA HlyIIC-15. MA number 15 (HlyIIC-15) was found in the panel of monoclonal antibodies against HlyIICTD, which is unable to recognize the antigen at the time of its conversation with the membranes of both erythrocytes [3] and liposomes [7]. The inability of HlyIIC-15 to interact with CTD bound to erythrocytes is usually apparently determined by the steric location of the epitope for this antibody and its inaccessibility relative to the membrane recognition point by the antigen. Miles et al. [8] exhibited that native hemolysin II and a deletion variant lacking the C-terminal domain name effectively oligomerize both in the presence of erythrocytes and liposomes. In this regard, the possibility of CTD oligomerization in the presence of erythrocytes and liposomes was tested. Figure 2 shows MA immunoblotting HlyIIC-15. As can be seen from this physique, HlyIICTD in the presence of Lerociclib dihydrochloride rabbit erythrocytes and liposomes transforms into a tetrameric form. Before the addition of erythrocytes and liposomes, in addition to monomeric forms of HlyIICTD, weakly colored dimeric and tetrameric forms of HlyIICTD are visible (Fig. 2, lane and sequenced peptides B771 cloned in BD170 (Table 2), was used. In Fig. 3, it is shown that MA HlyIIC-15 is able to form an immune complex with samples made up of a thrombin site and additional six histidine residues, but does not form such complexes with full-length wild-type HlyII, which does not contain either a thrombin site or six histidine amino acid residues. Thus, for the formation of an immune complex, the test antibody with the C-terminal region of HlyII in the recombinant protein, additional regions are required, including six histidine residues, a peptide linker, and a site recognized by thrombin..