Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

in Protein Expression & Purification (2012), 83(2), 198-204

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their ... [more ▼]

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella beta-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-beta-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination. [less ▲]

Purification of the recombinant beta2 toxin (CPB2) from an enterotoxaemic bovine Clostridium perfringens strain and production of a specific immune serum

in Protein Expression & Purification (2007), 55(1), 119-131

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The ... [more ▼]

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The so-called beta2 toxin (CPB2) is the most recently described major toxin produced by C perfringens. In this study, the cpb2 ORF (cpb2FM) from a cattle C perfringens-associated enterotoxaemia was cloned and sequenced. The cpb2FM and its deduced nucleotide sequence clearly corresponded to the epb2 allele considered as "consensus" and not to "atypical" allele, despite its "non-porcine" origin. Expression assays of the recombinant toxin CPB2FM were performed in Escherichia coli and Bacillus subtilis with the expression vector pBLTS72, and by genomic integration by double recombination in B. subtilis. Highest level of production was obtained with the expression vector in B. subtilis 168 strain. The recombinant CPB2FM protein was purified and a specific rabbit polyclonal antiserum was produced. Polyclonal antibodies could detect CPB2 production in supernatants of C. perfringens from enterotoxaemic cattle. (C) 2007 Elsevier Inc. All rights reserved. [less ▲]

Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.

in Protein Expression & Purification (1999), 16

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 ... [more ▼]

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoidingin vitroactivation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate α-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified. [less ▲]

Purification of Progelatinases A and B by Continuous-Elution Electrophoresis

in Protein Expression & Purification (1995), 6(4), 417-22

Progelatinases A and B were purified from HT1080-conditioned culture medium using a continuous-elution electrophoresis. Initially cell culture medium was ammonium sulfate precipitated. The concentrated ... [more ▼]

Progelatinases A and B were purified from HT1080-conditioned culture medium using a continuous-elution electrophoresis. Initially cell culture medium was ammonium sulfate precipitated. The concentrated proteins were affinity chromatographed on gelatin-Sepharose column. The bound gelatinases were eluted with electrophoresis sample buffer and subjected to continuous-elution electrophoresis. In a single run, under the standardized working conditions, the obtained fractions contained four purified enzymes--progelatinase A (M(r) 72,000), its activated forms (M(r) 62,000 and M(r) 59,000), and progelatinase B (M(r) 92,000). Moreover, the continuous-elution electrophoresis allowed the enzymes separation from their respective inhibitors--TIMP-1 (M(r) 28,500) and TIMP-2 (M(r) 21,000). The purified progelatinases A and B demonstrated high specific activities (150-200 U/micrograms). [less ▲]