On-column refolding and off-column assembly of parvovirus B19 virus-like particles from bacteria-expressed protein.

Virus-like particles (VLPs) are nanometric structures composed of structural components of virions, keeping most of the cellular recognition and internalization properties, but are non-infective as they are deprived of their genetic material. VLPs have been a versatile platform for developing vaccines by carrying their own or heterologous antigenic epitopes. Moreover, VLPs can also be used as nanovessels for encapsulating molecules with therapeutic applications, like enzymes, nucleic acids, and drugs. Parvovirus B19 (B19V) VLPs can be self-assembled in vitro from the denatured major viral particle protein VP2 by equilibrium dialysis. Despite its fair productivity, this process is currently a time-consuming task. Affinity chromatography is used as an efficient step for concentration and purification, but it is only sometimes seen as a method that facilitates the oligomerization of proteins. In this research, we report a novel approach for the in vitro assembly of B19V VLPs through the immobilization of the denatured VP2 into an immobilized metal affinity chromatography (IMAC) column, followed by the on-column folding and the final VLP assembly upon protein elution. This method is suitable for the fast production of B19V VLPs. KEY POINTS: • Biotechnological applications for inclusion bodies • Efficient single-step purification and immobilization strategies • Rapid VLP assembly strategy.

Therapeutic Potential of Engineered Virus-like Particles of Parvovirus B19.

Virus-like particles (VLPs) comprise one or many structural components of virions, except their genetic material. Thus, VLPs keep their structural properties of cellular recognition while being non-infectious. VLPs of Parvovirus B19 (B19V) can be produced by the heterologous expression of their structural proteins VP1 and VP2 in bacteria. These proteins are purified under denaturing conditions, refolded, and assembled into VLPs. Moreover, chimeric forms of VP2 have been constructed to harbor peptides or functional proteins on the surface of the particles without dropping their competence to form VLPs, serving as presenting nanoparticles. The in-vitro assembly approach offers exciting possibilities for the composition of VLPs, as more than one chimeric form of VP2 can be included in the assembly stage, producing multifunctional VLPs. Here, the heterologous expression and in-vitro assembly of B19V structural proteins and their chimeras are reviewed. Considerations for the engineering of the structural proteins of B19V are also discussed. Finally, the construction of multifunctional VLPs and their future potential as innovative medical tools are examined.

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 23 + 4 were performed to evaluate the following parameters: molar mass of PEG (MPEG ), concentration of PEG (CPEG ), concentration of citrate buffer (CCit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of MPEG  = 10,000 g/mol, CPEG  = 22 wt%, CCit  = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 µmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 µmol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUT s Strain.

L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUT s under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L-1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.g glycerol - 1 and a specific growth rate of 0.21 h-1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L-1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L-1, periplasmic specific enzyme activity 37.1 U.g dcw - 1 , volumetric enzyme activity 3,315 U.L-1, overall enzyme volumetric productivity 31 U.L-1.h-1, while the specific growth rate fell to 0.039 h-1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.