Development Of Purification Protocol Research Articles

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

Extracellular expression and affinity purification of L-asparaginase from E. chrysanthemi in E. coli

BackgroundL-asparaginases (E.C.3.5.1.1, L-ASNases) are successfully used as anti-neoplastic agents in the chemotherapy of acute lymphoblastic leukemia (ALL) and therefore are of high interest for the medical and scientific community. In the present study we report the recombinant, extracellular expression and affinity purification of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in E. coli.ResultsRecombinant E. coli strains were screened for their ability to express and translocate ErL-ASNase to the culture medium. The strain E. coli Rosetta (DE3) exhibited the highest extracellular expression levels among all the strains tested and it was chosen for further optimization and the development of purification protocol. Affinity adsorbents with immobilized L-Asn, L-Asp and L-Glu were synthesized by solid-phase chemistry and evaluated for their ability to bind and purify ErL-ASNase directly from the culture medium. The affinity adsorbent with immobilized L-Asp (L-Asp-Sepharose CL-6B) showed the highest purifying ability for ErL-ASNase. Adsorption equilibrium studies revealed that the adsorption of ErL-ASNase follows Langmuir isotherm with KD = 0.21 μM and maximum binding capacity 4.7 mg enzyme/g moist wet adsorbent. This affinity adsorbent was used for the development of one-step purification protocol. The proposed protocol is simple, fast, gentle and afforded ErL-ASNase with high purity and yield.ConclusionsWe show that the recombinant expression of ErL-ASNase in E. coli results in the secretion to the culture medium due to the presence of its natural peptide leader at the N-terminus. We developed an L-Asp-based affinity adsorbent which allowed the purification of the enzyme in one step, achieving high purity levels. This approach is advantageous over the other conventional tag-based purification methods, which require additional treatment steps for the cleavage and isolation of the affinity tags. Overall, the strategy employed for expression and purification of this protein drug uses green chemistry principles allowing the reduction of processing time and purification steps, making the approach more sustainable and attractive.

Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract -- critical parameters of protein isolation from anaerobic culture.

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-β-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.