Green fluorescent protein (GFP) has excellent properties as a biosensor and biomarker; however, its widespread use is limited by its purification costs. Alternative low-cost purification techniques can overcome this issue. The aim of this work was to evaluate aqueous biphasic systems (ABS) composed of cholinium chloride ([Ch]Cl) and different polymers as effective platforms to recover GFP from cell lysate of recombinant Escherichia coli BL21. All systems completely extracted GFP from cell lysate (>99%) into the polymeric-or [Ch]Cl-rich phases. In general, [Ch]Clbased ABS allowed a good purification capacity (GFP 80− 100% pure), with the best results (approximately 100% pure GFP) achieved with a polypropylene glycol (PPG)-400/[Ch]Cl ABS in a single-step extraction or in a two-step extraction (backextraction) by the integration of a polyethylene glycol (PEG)/sodium polyacrylate+[Ch]Cl ABS with a following stage using a PEG/[Ch]Cl-based ABS.Additionally, to demonstrate the potential of the PPG-400/[Ch]Cl ABS in downstream processing, solvent recycling and GFP polishing were carried out using ultrafiltration. Finally, the capacity of the PPG-400/[Ch]Cl ABS to extract other fluorescent proteins was also confirmed. The results clearly demonstrated that the PPG-400/[Ch]Cl ABS can be applied in downstream processing for the purification of proteins, not only enhancing purification yields but also providing simpler, quicker, cost-effective, and biocompatible processes.

Biopharmaceuticals are one of the most important groups of biotechnological products, representing one-quarter of all pharmaceutical sales and providing suitable and efficient medical care for many previously untreatable diseases. However, their production and purification usually require complex processes, resulting in extremely expensive final products. Thus, the development of newer, simpler and cheaper methods for biosynthesis and purification of these biocompounds, as well as proper integration between these stages, are crucial to reduce their commercial costs and to increase the scale of biopharmaceuticals' medical use. One solution for this concern relies on the proper integration between upstream and downstream processing applying liquid-liquid extraction systems as an intermediate stage. Recently, several works reported that a proper choice of liquid-liquid systems such as aqueous biphasic systems (ABS) can be a suitable platform to simplify, reduce the number of the downstream stages, improve the recovery and purification yields and, consequently, decrease biopharmaceuticals' manufacturing costs. This review will explain the general concept of biopharmaceuticals, their main production and purification methods, particularly, exploring the use of ABS as effective and integrative platforms for their recovery and purification, and providing further insights into the future trends and prospects in the field.

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f Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus ⌬mfp::kan and ⌬omp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a ⌬mfp::kan or ⌬omp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus ⌬omp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the ⌬mfp::kan strain had a lower virulence in these same mouse models. Furthermore, ⌬mfp::kan and ⌬omp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the ⌬mfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a ⌬omp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the ⌬mfp::kan mutant may serve as a potential vaccine candidate in future studies. Brucellosis is a chronic infectious disease caused by bacteria of the genus Brucella. This disease affects many species of animals, resulting in great economic losses, and is therefore an important bacterial zoonotic disease worldwide (1). The genus Brucella replicates inside trophoblasts, macrophages, and dendritic cells and colonizes the reticuloendothelial system and reproductive organs (2). Additionally, brucellosis is not only the major cause of abortion and infertility in animals but also a debilitating disease in humans (2-7).To overcome the immune system and establish a chronic infection, B. abortus utilizes diverse evasion mechanisms. This pathogen can penetrate host cells through lipid rafts (8). Once inside cells, the establishment of a persistent infection relies on the ability of the bacterium to form a Brucella-containing vacuole (BCV), which traffics from the endocytic compartment to the endoplasmic reticulum (ER), forming a replicative BCV. It is in this replicative BCV that the bacteria begin to multiply (8, 9).Extensive vaccination programs have been undertaken to prevent brucellosis in animals. Despite their availability, live vaccine strains have critical disadvantages (10). The main vaccines currently available for brucellosis are S19 and RB51 (derived from B. abortus)...

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