Establishment of an HPLC-based method to identify key proteases of proteins in vitro.

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 µm(-1) s(-1), optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (ß-propeller domain); and residues Ala(496)-Phe(736) (α/ß-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.

Localized gene expression in Pseudomonas aeruginosa biofilms.

Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-beta-D-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 microm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-microm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton.

The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.