Subcellular location of the coupling protein TrwB and the role of its transmembrane domain.

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid.

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein.

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim.

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate.

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB.

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDeltaN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBDeltaN70 binds uniformly all tested nucleotides.

Purification of different lipases from Aspergillus niger by using a highly selective adsorption on hydrophobic supports.

In this manuscript, we have purified three different lipases from crude preparations from Aspergillus niger in a simple fashion, secluding the esterases and other enzymes presented in the preparation. Firstly, the crude was offered at low ionic strength to octyl agarose. The support specifically adsorbed two lipases, with molecular weights of 43 and 65 kDa.

Determination of protein-protein interactions through aldehyde-dextran intermolecular cross-linking.

A very simple strategy, based on the intermolecular cross-linking of associated proteins by using aldehyde-dextrans, has been proposed to detect protein-protein interactions. Aldehyde-dextran was able to cross-link different enzymes composed of several polypeptide chains (e.g.

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7.

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts.

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups.