Dual pulsed laser deposition of Ag nanoparticles on calcium phosphate coatings for biomedical applications.

Pulsed laser deposition (PLD) represents a promising bottom-up methodology for the synthesis and transference of nanoparticles to the surface of a biomedical device. Silver (Ag) nanoparticles directly incorporated on the metallic implant emerge as an alternative strategy for local action against prosthetic joint-associated infections. In the present research, a dual sequential PLD process is proposed to obtain a bilayer coating with (1) a bio-derived calcium phosphate (CaP) layer, to provide osteointegrative properties and (2) the controlled growth of the Ag nanoparticles over it, ranging the number of laser pulses from 100 to 500. The characterization by SEM, EDS, TEM, XPS and AFM revealed the uniform deposit of Ag rounded nanoparticles, with a narrow mean size distribution, in the original non-oxidized metallic state. Moreover, given the evidences from XPS and AFM techniques, the occurrence of a coalescence phenomenon from 400 pulses onwards was proposed together with the expected positive linear relation between the number of pulses and Ag contribution with a deposition rate of 0.05 at. % of Ag per pulse. Conversely, the decrease in roughness as the Ag content increased was also verified. Finally, the expected bacteriostatic activity for these PLD deposited metallic state Ag nanoparticles against the bacterial strainStaphylococcus aureuswas confirmed. Moreover, the evaluation of the osteoblast-like MG-63 cells viability on the Ag(100-500)-CaP coatings revealed a significant increased proliferation (p > 0.05) on the Ag100-CaP coating compared to the control (Ag0-CaP). When same coating was evaluated againstS. aureusthe effect was not significant. The possibility of modulating the amount of nanoparticles in the bilayer coating to obtain a greater or lesser effect in combination with CaP was revealed.

Recombinant protein secretion in Escherichia coli.

The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.

Evaluation of bottlenecks in proinsulin secretion by Escherichia coli.

This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g(-1) dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17-24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle.

Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB.

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.