The interaction of TraW and TrbC is required to facilitate conjugation in F-like plasmids.

Bacterial conjugation, such as that mediated by the E. coli F plasmid, is a main mechanism driving bacterial evolution. Two important proteins required for F-pilus assembly and DNA transfer proficiency are TraW and TrbC. As members of a larger complex, these proteins assemble into a type IV secretion system and are essential components of pore formation and mating pair stabilization between the donor and the recipient cells. In the current report, we demonstrate the physical interaction of TraW and TrbC, show that TraW preferentially interacts with the N-terminal domain of TrbC, and that this interaction is important in restoring conjugation in traW/trbC knockouts.

Aptamer facilitated purification of functional proteins.

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.

Simultaneous Analysis of a Non-Lipidated Protein and Its Lipidated Counterpart: Enabling Quantitative Investigation of Protein Lipidation's Impact on Cellular Regulation.

Here, we introduce protein-lipidation quantitation (PLQ)-the first method for quantitative analysis of both a substrate and a product of protein lipidation in a biologically relevant context. Such analysis is required to study roles of protein lipidation in cellular regulation. In PLQ, the substrate is fused with a fluorescent protein to facilitate quantitative detection of both the nonlipidated substrate and the lipidated product. When expressed in cells with endogenous lipidation activity, the substrate is intracellularly lipidated. Following cell lysis and sampling crude cell lysate for analysis, the substrate and the product are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. In this work, we prove PLQ in principle and demonstrate its robustness to changes in structures of the substrate and lipid donor. Finally, PLQ analysis confirms a hypothesized link between a mutation in p53 and cellular prenylation activity.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard.

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment.