Polystyrene adsorbents: rapid and efficient surrogate for dialysis in membrane protein purification

Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction. Purifying functionally active form of membrane protein in sufficient quantity is a major bottleneck in establishing its structure and understanding the functional mechanism. Although overexpression of the membrane proteins has been achieved by recombinant DNA technology, a majority of the protein remains insoluble as inclusion bodies, which is extracted by detergents. Detergent removal is essential for retaining protein structure, function, and subsequent purification techniques. In this study, we have proposed a new approach for detergent removal from the solubilized extract of a recombinant membrane protein: human phospholipid scramblase 3 (hPLSCR3). N-lauryl sarcosine (NLS) has been established as an effective detergent to extract the functionally active recombinant 6X-his- hPLSCR3 from the inclusion bodies. NLS removal before affinity-based purification is essential as the detergent interferes with the matrix binding. Detergent removal by adsorption onto hydrophobic polystyrene beads has been methodically studied and established that the current approach was 10 times faster than the conventional dialysis method. The study established the potency of polystyrene-based beads as a convenient, efficient, and alternate tool to dialysis in detergent removal without significantly altering the structure and function of the membrane protein.

Production and characterization of bacteriocin produced by lactic acid bacteria isolated from rusip

Research was conducted to produce and characterize bacteriocin produced by lactic acid bacteria (LAB) isolated from rusip, a traditional Bangkanese fermented fish product. Experiment was started by isolation of lactic acid bacteria from rusip, followed by screening to obtain the best isolate which has the highest bacterial inhibition activity. The selected isolate was then identified and used to produce crude bacteriocin. The crude bacteriocin was characterized through its stability in high temperature and proteolytic enzymes, inhibitory spectrum, pH sensitivity and effect of surfactants. The result showed that CN1.10a isolate which was identified as Lactococcus lactis subsp lactis has the highest bacterial inhibition activities; therefore it was selected to produce crude bacteriocin. The bacteriocin produced was heat stable, sensitive to proteolytic enzymes i.e. proteinase-K and papain but not to RNase. It inhibited Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum. It stable at pH 2.0 to 6.0. Among surfactans used sodium dodecyl sulphate (SDS), lauryl sarcosine and EDTA were able to stimulate bacteriocin production, while the production were strongly inhibited by Tween 20, Tween 80, Triton X-100 and urea. Based on the above characteristic, the bacteriocin was suitable to be used as a preservative of food which has to be processed at high temperature.

Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC

ABSTRACT The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 μg/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 μM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.Key words: triplex DNA, pulsed-field gel electrophoresis, chromosome structure, DNA mobility, chromosome condensation.