Cloning and expression of the alkaline proteinase gene from Pseudomonas aeruginosa IFO 3455

Abstract

The alkaline proteinase gene from Pseudomonas Escherichia coli.aeruginosa IFO 3455 was cloned and expressed inThe roles of several extracellular products have been established or implicated in the pathogenicity of Pseudomonas aeruginosa. These include proteases, phospholipase, hemolysin, exotoxin A, and exoenzyme S (6,8). Protease pathogenicity is explained by an aggressin activity (7), whereby the virulence of non-protease-producing strains is increased by the administration of a minute amount of protease to an experimental animal infected with P. aeruginosa. P. aeruginosa can produce two or three proteases (5), one of which is alkaline proteinase. This enzyme was crystallized from fermentation broth of strain PM and from that of IFO 3080. Alkaline proteinase can be regarded as a metalloproteinase, since it is inactivated by the addition of o-phenanthroline (7,8). In the present study we were able to clone the alkaline proteinase gene; the characterization of the cloned gene, as well as its restriction map, is also given.P. aeruginosa IFO 3455 and IFO 3080 were obtained from the Institute for Fermentation, Osaka, Japan. Crystal alkaline proteinase of P. aeruginosa IFO 3080 was obtained from Nagase Biochemical Co., Kyoto, Japan. Anti-alkaline proteinase antibody was produced in rabbits by injecting 0.4 mg of alkaline proteinase, which had been 80% inactivated by Formalin, per animal. The anti-alkaline proteinase antibody was detectable at 32x by Ouchterlony assay.As a first step, we constructed the SphI-digested genomic libraries from P. aeruginosa IFO 3455. The libraries were screened with rabbit anti-alkaline proteinase antibody and then treated with peroxidase-labeled anti-rabbit immunoglobulin antibody. By using this procedure, several clones were obtained out of about 6,000 possible clones. However, when the antibody assays were redone, only one clone [Escherichia coli HB101(pAP101)] reacted repeatedly. This pAP101 plasmid contained a fragment of P. aeruginosa DNA of approximately 5.3 kilobases (kb). From this clone, we constructed the subclone pAPDS2 by unidirectional digestion with exonuclease III (2, 3, 9); this subclone possessed both the antigenicity and enzyme activity of alkaline proteinase. Figure 1 shows the restriction enzyme map of the cloned gene.The alkaline proteinase activity of each clone was studied. E. coli HB101(pAP101), E. coli HB101(pAPDS2), E. coli HB101(pUC18), and parental E. coli HB101 were cultured for 24 h at 37°C in L broth. The bacteria were washed, and these suspensions were sonicated and then ultracentrifuged at 42,000 x g for 10 min. The supernatant fractions were * Corresponding author. assayed to determine enzyme activity as the soluble fraction ( Table 1). The precipitants were solubilized with 8 M urea. Solubilized samples of 42,000 x g precipitants were again ultracentrifuged and dialyzed against 10 mM Tris buffer (pH 8.0). Only these supernatants were used as the bacterial precipitate fractions. Alkaline proteinase activity was determined by a modification of the method...

Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

“…The gene for the full-length mature form of AprA [24-26] which corresponds to residues G10 to V479 was isolated on to pET22b and overexpressed in E. coli BL21 (λDE3), resulting in inclusion body formation. Isolation of the protein aggregates followed by solubilisation and refolding produced an active, stable and soluble enzyme of 49 500 Da, according to mass spectrometric analysis (Figure 2 and 3).…”

“…When the structural gene for AprA was expressed alone, however, intracellular accumulation of AprA was barely detectable, most likely due to rapid degradation inside the cell [52]. In other reports, significant quantities of the enzyme could be found intracellularly, but only in the form of inclusion bodies, which are known to be highly resistant to degradation [24,53,54]. …”

Partial Characterization and Cloning of Protease From Bacillus

“…The present state of knowledge of protein export in Bacillus species is still relatively poor. Therefore, the most thoroughly characterized export systems of E. coli [19] are utilized for the expression of industrially important enzymes such as alkaline proteases. The recombinants of E. coli harboring the protease gene from Erwinia chrysanthemi and from Pseudomonas aeurginosa [20] have been shown to secrete recombinant proteins.…”

Quantitation and Enzymatic Activity of the Alkaline Protease from Pseudomonas aeruginosa in Culture Supernatants from Clinical Strains

“…We have compared both concentrations and activities of AP corresponding to the different strains (Fig. It is known that alkaline protease may be present in supernatants in two forms: one of 49.5 kDa, which corresponds to the active enzyme and another one of 51 kDa, which is supposed to be an inactive precursor (14). A statistic study was realised on the basis of a linear relationship 296 Louis et al: Alkaline protease from Pseudomonas aeruginosa: quantitation and activity between activity and concentration.…”